Peptide libraries

ABSTRACT

The invention relates to a method for altering the conformational diversity of a first repertoire of polypeptide ligands, comprising a plurality of polypeptides comprising at least two reactive groups separated by a loop sequence covalently linked to a molecular scaffold which forms covalent bonds with said reactive groups, to produce a second repertoire of polypeptide ligands, comprising assembling said second repertoire from the polypeptides and structural scaffold of said first repertoire, incorporating one of the following alterations: (a) altering at least one reactive group; or (b) altering the nature of the molecular scaffold; or (c) altering the bond between at least one reactive group and the molecular scaffold; or (d) any combination of (a), (b) or (c).

The present application is a continuation of U.S. Ser. No. 13/390,252filed Mar. 14, 2012, which is a filing under 35 USC §371 ofPCT/EP2010/004948 filed Aug. 12, 2010, which claims priority to GB0914110.2 filed Aug. 12, 2009.

The present invention relates to peptides whose structure is constrainedby binding to a compound which provides a structural backbone, impartinga conformation to the peptide. In particular, the invention relates tomodifying the conformational diversity of libraries of such peptides byaltering the interaction between the peptides and the structuralbackbone.

Different research teams have previously tethered polypeptides withcysteine residues to a synthetic molecular structure (Kemp, D. S. andMcNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., Chem BioChem, 2005). Meloen and co-workers had used tris(bromomethyl)benzene andrelated molecules for rapid and quantitative cyclisation of multiplepeptide loops onto synthetic scaffolds for structural mimicry of proteinsurfaces (Timmerman, P. et al., Chem Bio Chem, 2005). Methods for thegeneration of candidate drug compounds wherein said compounds aregenerated by linking cysteine containing polypeptides to a molecularscaffold as for example tris(bromomethyl)benzene are disclosed in WO2004/077062 and WO 2006/078161.

WO2004/077062 discloses a method of selecting a candidate drug compound.In particular, this document discloses various scaffold moleculescomprising first and second reactive groups, and contacting saidscaffold with a further molecule to form at least two linkages betweenthe scaffold and the further molecule in a coupling reaction.

WO2006/078161 discloses binding compounds, immunogenic compounds andpeptidomimetics. This document discloses the artificial synthesis ofvarious collections of peptides taken from existing proteins. Thesepeptides are then combined with a constant synthetic peptide having someamino acid changes introduced in order to produce combinatoriallibraries. By introducing this diversity via the chemical linkage toseparate peptides featuring various amino acid changes, an increasedopportunity to find the desired binding activity is provided. FIG. 7 ofthis document shows a schematic representation of the synthesis ofvarious loop peptide constructs. The constructs disclosed in thisdocument rely on —SH functionalised peptides, typically comprisingcysteine residues, and heteroaromatic groups on the scaffold, typicallycomprising benzylic halogen substituents such as bis- ortris-bromophenylbenzene. Such groups react to form a thioether linkagebetween the peptide and the scaffold.

In our copending unpublished international patent applicationPCT/GB2009/000301 we disclose the use of biological selectiontechnology, such as phage display, to select peptides tethered tosynthetic molecular structures.

SUMMARY OF THE INVENTION

The nature of the interaction between the scaffold and the polypeptideis important in determining the structural form of the peptide-scaffoldconjugate. Although the prior art exploits scaffolds to impart structureto polypeptides, and therefore allows the introduction of conformationaldiversity through alteration of the sequence of the peptides, it doesnot recognise that structure as well as structural diversity can bealtered by manipulating the interaction between the peptide and thescaffold, as well as changing the scaffold itself.

The present invention provides for the alteration of structure and/orthe introduction of structural diversity by means of the scaffold andthe scaffold-peptide interface.

In a first aspect, therefore, there is provided a method for alteringthe conformation of a first polypeptide ligand or group of polypeptideligands, each polypeptide ligand comprising at least two reactive groupsseparated by a loop sequence covalently linked to a molecular scaffoldwhich forms covalent bonds with said reactive groups, to produce asecond polypeptide ligand or group of polypeptide ligands, comprisingassembling said second ligand or group of ligands from thepolypeptide(s) and scaffold of said first ligand or group of ligands,incorporating one of:

-   -   a) altering at least one reactive group; or    -   b) altering the nature of the molecular scaffold; or    -   c) altering the bond between at least one reactive group and the        molecular scaffold; or    -   d) any combination of (a), (b) or (c).

The approaches adopted in the present invention allow the person skilledin the art to modify the conformation of any structured polypeptidecomprising a polypeptide covalently linked to a molecular scaffold, aswell as groups of such peptides. Altering the conformation of astructured polypeptide will affect its function. For example, where thestructured polypeptide is a binding molecule, the binding specificityand/or the binding affinity may be modified. Binding affinity may beincreased, or decreased. Increases in affinity can have advantages, forinstance in allowing lower quantities of a therapeutic or diagnosticagent to be used. Likewise, compounds with reduced affinity may bepreferred, for example to reduce background from low levels of targetproteins, as T-cell receptor ligands, and the like.

Alterations in the reactive group typically comprise changes in theamino acid side chain responsible for forming the covalent bond with thescaffold. For example the reactive amine or thiol group can besubstituted. This can take place by amino acid replacement, for instancethe replacement of cysteine with another natural amino acid, such aslysine; replacement with an alternative amino acid, such asselenocysteine or azido phenylalanine; and modification of theenvironment in which the reactive group is situated in the polypeptide,for example by changing one or both of the adjacent amino acids, thusinfluencing the peptide structure.

Altering the nature of the molecular scaffold includes, for instance,changing the nature of the scaffold reactive group. For example,altering a tris(bromomethyl)benzene scaffold to atris(bromomethyl)mesitylene scaffold introduce methyl groups attached tothe benzene ring, and allows a lesser degree of “play” between thescaffold and the polypeptide, leading to a more rigid structure. If thestructure of the polypeptide conjugate is complementary to a target,then the binding of the rigid structure to the target may be favouredover that of a more flexible structure; on the other hand if thestructure of the polypeptide conjugate is not complementary, the bindingof the more flexible structure to the target may be preferred, as thereis more possibility for an adaptive fit between the peptide ligand and atarget. A number of alternative scaffold reactive groups are available,as set forth below, and many more will be apparent to those skilled inthe art.

The scaffold itself may also be altered, for instance by selecting adifferent molecular structure. This may have a degree of structuralresemblance to the scaffold in the first repertoire, or not; thus, theinvention contemplates using a structurally similar, but chemicallydifferent scaffold. Moreover, the invention comprises using astructurally different scaffold.

Altering the bond between a reactive group and the scaffold can resultfrom alterations in the nature of a reactive group or a scaffoldreactive group. Moreover, it can be a result of modification of the bondpost-attachment of the scaffold. For example, oxidation of thioetherlinkages between the scaffold and the peptide leads to the formation ofsulphoxides, which have an altered geometry and therefore impart adifferent structure to the polypeptide in the peptide ligand.

Alterations introduced to the scaffold, polypeptide or the bonds neednot be symmetrical. For example, if there are two or three reactivegroups on the peptide, only one of them need be altered to introduceincreased library diversity. Alternatively, a percentage of the reactivegroups can be altered; for example, 20, 30, 40 or 50% of the polypeptidemolecules can be constructed with altered reactive groups in one or morepositions, leaving the remaining polypeptides unmodified or modified ina different way.

Scaffold alterations, likewise, need not be symmetrical. An asymmetricalscaffold may present different scaffold reactive groups in one, two,three or more positions. Such groups may be orthogonal, dictating aparticular arrangement of binding with the polypeptide, thus reducingthe number of structural isomers of the peptide ligand which can beformed. Alternatively, the formation of isomers may be allowed, topromote even greater diversity.

Assembly of the libraries of the invention can itself be exploited tointroduce diversity. For instance, a repertoire of polypeptides may beexposed to two or more scaffold species, and possibly a repertoire ofscaffolds, leading to increased diversity through differing combinationsof sequence variation and scaffold variations. If a scaffold has threescaffold reactive groups, a significant degree of variation can beintroduced merely by randomising the nature of said scaffold reactivegroups. Combined with the randomisation of the polypeptide sequence,this can lead to greatly increased repertoire size.

When applied to repertoires of polypeptides, the methods according tothe present invention may be used to increase the structural diversityof polypeptide ligands. Moreover, further diversity can be obtained bychoosing a different scaffold, which may have differing structuralproperties.

In a preferred embodiment, the invention comprises the generation of agroup or repertoire of polypeptide ligands from smaller group ofpolypeptide ligands, including for example a single polypeptide ligand.In such embodiments, diversity is generated through any one or more ofalteration or replacement of the scaffold, alteration of at least onereactive group in the peptide, and/or altering at least one bond betweenthe polypeptide and the scaffold. Suitably, where necessary, thediversity of the repertoire may be further enhanced by modification ofthe sequence of the peptide. For example, amino acid additions,deletions or substitutions may be made between the reactive groups inthe polypeptide, therefore altering the sequence of the loops subtendedbetween the attachment points to the scaffold. For example, one loop maybe altered, and a second loop may be left unchanged.

Accordingly, the invention provides a method for generating a group ofdiverse polypeptide ligands from a first polypeptide ligand comprisingat least two reactive groups separated by a loop sequence covalentlylinked to a molecular scaffold which forms covalent bonds with saidreactive groups, comprising assembling a group or repertoire of ligandsfrom the polypeptide and scaffold of said first ligand or group ofligands, and incorporating one of the following alterations:

-   -   a) altering at least one reactive group; or    -   b) altering the nature of the molecular scaffold; or    -   c) altering the bond between at least one reactive group and the        molecular scaffold; or    -   d) any combination of (a), (b) or (c); or    -   e) modifying the sequence of the polypeptide, in combination        with any one of (a) to (d).

In one embodiment, diversity may be generated by subjecting one or moreloops of the polypeptide ligand to complete or partial proteolysis.Cleavage of one or more loops of the ligand can alter the structure andtherefore the functional properties thereof. Advantageously, proteolysiscan be used in conjunction with modification of the polypeptide sequenceof one or more of the loops.

In a second aspect, the invention provides a method for increasing theconformational diversity of a first repertoire of polypeptide ligands,comprising a plurality of polypeptides comprising at least two reactivegroups separated by a loop sequence covalently linked to a molecularscaffold which forms covalent bonds with said reactive groups,comprising assembling a second repertoire of peptide ligands from thepolypeptides of said first repertoire and at least two structurallydiverse molecular scaffold species. The structurally different molecularscaffold species may differ in molecular structure and/or the nature ofone or more scaffold reactive groups.

Variation of the molecular scaffold may be used to carry out affinitymaturation of selected polypeptide ligands. In one embodiment, there isprovided a method for providing a polypeptide ligand comprising apolypeptide covalently linked to a molecular scaffold at two or moreamino acid residues, comprising the steps of:

(a) providing a first repertoire of polypeptides;(b) conjugating said polypeptides to a molecular scaffold which binds tothe polypeptides at two or more amino acid residues, to form a firstrepertoire of polypeptide conjugates;(c) screening said first repertoire for binding against a target, andselecting members of the first repertoire which bind to the target;(d) introducing further variation into the polypeptide ligands, inaccordance with the first aspect of the invention set forth above,yielding a second repertoire of polypeptide conjugates; and(e) screening said second repertoire for improved binding to the target.

Alterations in the scaffold which increase or reduce the flexibility ofthe polypeptide structure, in particular, may have pronounced effects onbinding affinity without necessarily altering binding specificity.

Moreover, the invention provides a method for altering the bindingactivity of a polypeptide ligand comprising a polypeptide comprising atleast two reactive groups separated by a loop sequence covalently linkedto a molecular scaffold which forms covalent bonds with said reactivegroups at two or more amino acid residues, comprising the steps of:

-   -   (a) altering at least one reactive group; or    -   (b) altering the nature of the molecular scaffold; or    -   (c) altering the bond between at least one reactive group and        the molecular scaffold; or    -   (d) any combination of (a), (b) or (c); or    -   (e) modifying the sequence of the polypeptide, in combination        with any one of (a) to (d).

Screening may be carried out by analysis of individual molecules. Suchmethods are provided in WO 2004/077062 and WO 2006/078161. However,screening of individual compounds or small sets of compounds is tediousand can be expensive if large numbers of compounds are analyzed. Thenumber of compounds that can be assayed with screening assays generallydoes not exceed several thousands. Since screening of large numbers ofpolypeptide ligands in this way can be inefficient, an improvedscreening capability is highly desirable.

In a preferred embodiment, the repertoires of polypeptides are providedin the form of a nucleic acid library, and incorporated as part of agenetic display system. Applicable systems include phage display,bacterial display, yeast display, ribosome or polysome display, mRNAdisplay and in vitro expression in artificial microcapsules. Thepreferred technique is phage display using a filamentous bacteriophage.

The polypeptide ligand of the invention comprises at least onepolypeptide loop, subtended between two reactive groups on a molecularscaffold.

Preferably, the polypeptide conjugate of the invention comprises twoloops. It may specific for a single target, or multispecific, binding totwo or more targets. Several such polypeptide conjugates may beincorporated together into the same molecule. For example two suchpolypeptide conjugates of the same specificity can be linked togethervia the molecular scaffold, increasing the avidity of the ligand for itstargets. Alternatively, in another embodiment a plurality of polypeptideconjugates are combined to form a multimer. For example, two differentpolypeptide conjugates are combined to create a multispecific molecule.Alternatively, three or more polypeptide conjugates, which may be thesame or different, can be combined to form multispecific ligands.

In one embodiment multivalent complexes may be constructed by linkingtogether the molecular scaffolds, which may be the same or different.

One skilled in the art will appreciate that the choice of targetmolecule is large and varied. They may be for instance human or animalproteins, cytokines, cytokine receptors, enzymes co-factors for enzymesor DNA binding proteins. Suitable cytokines and growth factors includebut are not limited to: ApoE, Apo-SAA, BDNF, Cardiotrophin-1, EGF, EGFreceptor, ENA78, Eotaxin, Eotaxin-2, Exodus-2, FGF-acidic, FGF-basic,fibroblast growth factor-10 (30). FLT3 ligand, Fractalkine (CX3C), GDNF,G-CSF, GM-CSF, GF-I, insulin, IFNy, IGF-I, IGF-II, IL-la, IL-1 (3, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 (72 a.a.), IL-8 (77 a.a.), IL-9,IL-10, IL-11, IL-12, IL-13, IL-15, IL-16, IL-17, IL-17a, IL-17c, IL-17d,IL-17e, IL-17f, IL-18 (IGIF), IL-21, IL-22, IL-23, IL-31, IL-32, IL-33,IL-34, Inhibin α, Inhibin β, IP-10, keratinocyte growth factor-2(KGF-2), KGF, Leptin, LIF, Lymphotactin, Mullerian inhibitory substance,monocyte colony inhibitory factor, monocyte attractant protein (30ibid), M-CSF, MDC (67 a. a.), MDC (69 a. a.), MCP-1 (MCAF), MCP-2,MCP-3, MCP-4, MDC (67 a. a.), MDC (69 a. a.), MIG, MIP-Ia, MIP-1p,MIP-3a, MIP3 (3, MIP-4, myeloid progenitor inhibitor factor-1 (MPIF-1),NAP-2, Neurturin, Nerve growth factor, P-NGF, NT-3, NT-4, Oncostatin M,PDGF-AA, PDGF-AB, PDGF-BB, PF-4, RANTES, SDFIa, SDFIp, SCF, SCGF, stemcell factor (SCF), TARC, TGF-α, TGF-β, TGF-2, TGF-3, tumour necrosisfactor (TNF), TNF-α, TNF-β, TNF receptor I, TNF receptor II, TNIL-1,TPO, VEGF, VEGF receptor 1, VEGF receptor 2, VEGF receptor 3, GCP-2,GRO/MGSA, GRO-β, GRO-γ, HCC1,1-309, HER 1, HER 2, HER 3 and HER 4.Cytokine receptors include receptors for the foregoing cytokines.Chemokine targets include CC chemokine ligands CCL21/6Ckine,CCL12/MCP-5, CCL6/C10, CCL22/MDC, CCL14/HCC-1/HCC-3, CCL3L1/MIP-1 alphaIsoform LD78 beta, CCL23/Ck beta 8-1, CCL3/MIP-1 alpha, CCL28,CCL4L1/LAG-1, CCL27/CTACK, CCL4/MIP-1 beta, CCL24/Eotaxin-2/MPIF-2,CCL15/MIP-1 delta, CCL26-like/Eotaxin-3-like, CCL9/10/MIP-1 gamma,CCL26/Eotaxin-3, CCL19/MIP-3 beta, CCL11/Eotaxin, CCL20/MIP-3 alpha,CCL14a/HCC-1, CCL23/MPIF-1, CCL14b/HCC-3, CCL18/PARC, CCL16/HCC-4,CCL5/RANTES, CCL1/1-309/TCA-3, TAFA1/FAM19A1, MCK-2, TAFA5/FAM19A5,CCL2/JE/MCP-1, TAFA3/FAM19A3, CCL8/MCP-2, TAFA4/FAM19A4,CCL7/MCP-3/MARC, CCL17/TARC, CCL13/MCP-4 and CCL25/TECK; chemokinereceptors include CCR1, CCR7, CCR2, CCR8, CCR3, CCR9, CCR4, CCR10, CCR5,CCRL2/LCCR/CRAM-A/B and CCR6; CXC chemokine ligands includeCXCL13/BLC/BCA-1, CXCL10/IP-10/CRG-2, CXCL14/BRAK, LIX, CXCL16,CXCL15/Lungkine, CXCL5/ENA-78, CXCL9/MIG, CXCL6/GCP-2, CXCL7/NAP-2,CXCL1/2/3/GRO, CXCL4/PF4, CXCL1/GRO alpha/KC/CINC-1, CXCL12/SDF-1 alpha,CXCL2/GRO beta/MIP-2/CINC-3, CXCL12/SDF-1 beta, CXCL3/GROgamma/CINC-2/DCIP-1, CXCL12/SDF-1, CXCL11/I-TAC, CXCL7/ThymusChemokine-1 and CXCL8/IL-8; CXC chemokine receptors include CXCR3,CXCR7/RDC-1, CXCR4, CXCR1/IL-8 RA, CXCR5, CXCR2/IL-8 RB and CXCR6; TNFSuperfamily ligands include 4-1BB Ligand/TNFSF9, LIGHT/TNFSF14,APRIL/TNFSF13, Lymphotoxin, BAFF/BLyS/TNFSF13B, Lymphotoxin beta/TNFSF3,CD27 Ligand/TNFSF7, OX40 Ligand/TNFSF4, CD30 Ligand/TNFSF8,TL1A/TNFSF15, CD40 Ligand/TNFSF5, TNF-alpha/TNFSF1A, EDA (pan),TNF-beta/TNFSF1B, EDA-A1/Ectodysplasin A1, TRAIL/TNFSF10, EDA-A2,TRANCE/TNFSF11, Fas Ligand/TNFSF6, TWEAK/TNFSF12 and GITRLigand/TNFSF18; TNF Superfamily receptors include 4-1BB/TNFRSF9/CD137,NGF R/TNFRSF16, BAFF R/TNFRSF13C, Osteoprotegerin/TNFRSF11B,BCMA/TNFRSF17, OX40/TNFRSF4, CD27/TNFRSF7, RANK/TNFRSF11A, CD30/TNFRSF8,RELT/TNFRSF19L, CD40/TNFRSF5, TACI/TNFRSF13B, DcR3/TNFRSF6B,TNFRH3/TNFRSF26, DcTRAIL R1/TNFRSF23, TNF RI/TNFRSF1A, DcTRAILR2/TNFRSF22, TNF RII/TNFRSF1B, DR3/TNFRSF25, TRAIL R1/TNFRSF10A,DR6/TNFRSF21, TRAIL R2/TNFRSF10B, EDAR, TRAIL R3/TNFRSF10C,Fas/TNFRSF6/CD95, TRAIL R4/TNFRSF10D, GITR/TNFRSF18, TROY/TNFRSF19,HVEM/TNFRSF14, TWEAK R/TNFRSF12, Lymphotoxin beta R/TNFRSF3 and XEDAR;Toll-Like Receptors including TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6,TLR-7, TLR-8 and TLR-9; enzymes, including Cathepsin A, Cathepsin B,Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin F, MMP 1, MMP2, MMP 3,MMP 7, MMP 8, MMP 9, MMP 10, MMP 11, MMP 12, MMP 13, MMP 14, MMP 15, MMP16, MMP 17, MMP 19, MMP 20, MMP 21, MMP 23A, MMP 23B, MMP 26, MMP 27,MMP 28, urokinase, kallikreins, including KLK1, KLK2, KLK3, KLK4, KLK5,KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14 and KLK15;components of the complement system; Intracellular signalling moleculesand transcription factors; p53; and MDM2.

Targets may also be large plasma proteins, such as serum albumins, asset forth below.

It will be appreciated that this list is by no means exhaustive.

In a third aspect, the present invention provides a polypeptideconjugate comprising a polypeptide comprising at least three reactivegroups each separated by a loop sequence, covalently linked to amolecular scaffold comprising at least three scaffold reactive groupswhich form covalent bonds with said reactive groups, wherein themolecular scaffold comprises at least two different scaffold reactivegroups.

Preferably, the molecular scaffold comprises three different reactivegroups.

Additional binding or functional activities may be attached to the N orC terminus of the peptide covalently linked to a molecular scaffold. Thefunctional group is, for example, selected from the group consisting of:a group capable of binding to a molecule which extends the half-life ofthe polypeptide ligand in vivo, and a molecule which extends thehalf-life of the polypeptide ligand in vivo. Such a molecule can be, forinstance, HSA or a cell matrix protein, and the group capable of bindingto a molecule which extends the half-life of the polypeptide ligand invivo is an antibody or antibody fragment specific for HSA or a cellmatrix protein.

In one embodiment, the functional group is a binding molecule, selectedfrom the group consisting of a second polypeptide ligand comprising apolypeptide covalently linked to a molecular scaffold, and an antibodyor antibody fragment. 2, 3, 4, 5 or more polypeptide ligands may bejoined together. The specificities of any two or more of these ligandsmay be the same or different; if they are the same, a multivalentbinding structure will be formed, which has increased avidity for thetarget compared to univalent binding molecules. The molecular scaffolds,moreover, may be the same or different, and may subtend the same ordifferent numbers of loops.

The functional group can moreover be an effector group, for example anantibody Fc region.

Attachments to the N or C terminus may be made prior to binding of thepeptide to a molecular scaffold, or afterwards. Thus, the peptide may beproduced (synthetically, or by expression of nucleic acid) with an N orC terminal polypeptide group already in place. Preferably, however, theaddition to the N or C terminus takes place after the peptide has beencombined with the molecular backbone to form a conjugate. For example,Fluorenylmethyloxycarbonyl chloride can be used to introduce the Fmocprotective group at the N-terminus of the polypeptide. Fmoc binds toserum albumins including HSA with high affinity, and Fmoc-Trp orFMOC-Lys bind with an increased affinity. The peptide can be synthesisedwith the Fmoc protecting group left on, and then coupled with thescaffold through the cysteines. An alternative is the palmitoyl moietywhich also binds HSA and has, for example been used in Liraglutide toextend the half-life of this GLP-1 analogue.

The Fmoc group confers human serum albumin binding function to thebicyclic peptide. Alternatively, a conjugate of the peptide with thescaffold can be made, and then modified at the N-terminus, for examplewith the amine- and sulfhydryl-reactive linkerN-e-maleimidocaproyloxy)succinimide ester (EMCS). Via this linker thepeptide conjugate can be linked to other polypeptides, for example anantibody Fc fragment.

The binding function may be another peptide bound to a molecularscaffold, creating a multimer; another binding protein, including anantibody or antibody fragment; or any other desired entity, includingserum albumin or an effector group, such as an antibody Fc region.

Additional binding or functional activities can moreover be bounddirectly to the molecular scaffold.

Advantageously, the molecular scaffold comprises a reactive group towhich the additional activities can be bound. Preferably, this group isorthogonal with respect to the other reactive groups on the molecularscaffold, to avoid interaction with the peptide. In one embodiment, thereactive group may be protected, and deprotected when necessary toconjugate the additional activities.

In another aspect, the invention further provides a kit comprising atleast a peptide ligand according to the present invention.

In a still further aspect, the present invention provides a compositioncomprising a peptide ligand, obtainable by a method of the presentinvention, and a pharmaceutically acceptable carrier, diluent orexcipient.

Moreover, the present invention provides a method for the treatment ofdisease using a peptide ligand or a composition according to the presentinvention.

In a further aspect, the present invention provides a method for thediagnosis, including diagnosis of disease using a peptide ligand, or acomposition according to the present invention. Thus in general thebinding of an analyte to a peptide ligand may be exploited to displacean agent, which leads to the generation of a signal on displacement. Forexample, binding of analyte (second target) can displace an enzyme(first target) bound to the peptide ligand providing the basis for abinding assay, especially if the enzyme is held to the peptide ligandthrough its active site.

A particular advantage of the polypeptide conjugates of the inventionare smaller than binding agents of the prior art. Typically, such aligand has a molecular weight of less than 5000 Dalton; preferably lessthan 4000 Dalton; and preferably less than 3000 Dalton. It will beunderstood that a ligand constructed by “daisy-chaining” peptide ligandsas described in the second configuration of the invention will possess ahigher molecular weight. Moreover, peptide ligands bound to moleculessuch as HSA will have a much higher molecular weight.

The small size of the ligands results from the use of small molecularscaffolds, typically 500 Dalton in mass. The peptide itself ispreferably less than 27 amino acids in length, as measured between theN-terminal and C-terminal attachment points which attach it to themolecular scaffold. Further peptides may, of course, be present or beattached outside of the attachment points, lengthening the peptidestructure. Each loop of the polypeptide is preferably between 0 and 9amino acids in length, measured between adjacent attachment points.Advantageously, the loops in any peptide ligand are independently 3, 4,5, 6, 7, 8 or 9 amino acids in length.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art, such as in the arts of peptide chemistry, cell culture andphage display, nucleic acid chemistry and biochemistry. Standardtechniques are used for molecular biology, genetic and biochemicalmethods (see Sambrook et al., Molecular Cloning: A Laboratory Manual,3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; Ausubel et al., Short Protocols in Molecular Biology (1999) 4^(th)ed., John Wiley & Sons, Inc.), which are incorporated herein byreference.

A peptide ligand, as referred to herein, refers to a peptide covalentlybound to a molecular scaffold. Typically, such peptides comprise two ormore reactive groups which are capable of forming covalent bonds to thescaffold, and a sequence subtended between said reactive groups which isreferred to as the loop sequence, since it forms a loop when the peptideis bound to the scaffold.

The reactive groups are groups capable of forming a covalent bond withthe molecular scaffold. Typically, the reactive groups are present onamino acid side chains on the peptide. Preferred are amino-containinggroups such as cysteine, lysine and selenocysteine.

A target is a molecule or part thereof to which the peptide ligandsbind. Typically, the target will be analogous to an epitope.

The molecular scaffold is any molecule which is able to connect thepeptide at multiple points to impart one or more structural features tothe peptide. It is not a cross-linker, in that it does not merelyreplace a disulphide bond; instead, it provides two or more attachmentpoints for the peptide. Preferably, the molecular scaffold comprises atleast three attachment points for the peptide, referred to as scaffoldreactive groups. These groups are capable of reacting to the reactivegroups on the peptide to form a covalent bond. Preferred structures formolecular scaffolds are described below.

A repertoire is a collection of variants, in this case polypeptidevariants, which differ in their sequence. In some embodiments, thelocation and nature of the reactive groups will not vary, but thesequences forming the loops between them can be randomised. In otherembodiments, the reactive groups themselves may vary. The size of arepertoire may vary, and in the present invention may depend on thepurpose of the repertoire. For example, the first repertoire isadvantageously a large repertoire, which may be suitable for selectionusing phage. Such repertoires advantageously consist of at least 10²,10³, 10⁴, 10⁵ or more different polypeptide variants. Smallerrepertoires may not be intended for selection by a genetic displaysystem, for example phage display, and might consist of 100 diversepolypeptide variants, or fewer. For example, they may consist of 10, 20,30 40 or 50 diverse polypeptide variants. For example, therefore,starting from a first repertoire, a subset of the members may be variedaccording to methods set forth herein, to produce a second, smallerrepertoire which can itself be screened without using a genetic displaytechnology such as phage display.

A group of polypeptide variants comprises at least two diversepolypeptide variants, and may comprise 5, 10, 15, 20, 25, 30, 40 50 ormore diverse variants. As with smaller repertoires, groups of variantscan be screened without using a genetic display technology such as phagedisplay, but are amenable to screening using any suitable polypeptidedisplay technology, such as solid phase polypeptide arrays, microtitreplates and the like.

Screening for binding activity (or any other desired activity) isconducted according to methods well known in the art, for instance fromphage display technology. For example, targets immobilised to a solidphase can be used to identify and isolate binding members of arepertoire. Screening allows selection of members of a repertoireaccording to desired characteristics.

The term library refers to a mixture of heterogeneous polypeptides ornucleic acids. The library is composed of members, each of which has asingle polypeptide or nucleic acid sequence. To this extent, library issynonymous with repertoire. Sequence differences between library membersare responsible for the diversity present in the library. The librarymay take the form of a simple mixture of polypeptides or nucleic acids,or may be in the form of organisms or cells, for example bacteria,viruses, animal or plant cells and the like, transformed with a libraryof nucleic acids. Preferably, each individual organism or cell containsonly one or a limited number of library members.

Advantageously, the nucleic acids are incorporated into expressionvectors, in order to allow expression of the polypeptides encoded by thenucleic acids. In a preferred aspect, therefore, a library may take theform of a population of host organisms, each organism containing one ormore copies of an expression vector containing a single member of thelibrary in nucleic acid form which can be expressed to produce itscorresponding polypeptide member. Thus, the population of host organismshas the potential to encode a large repertoire of genetically diversepolypeptide variants.

Preferably, a library of nucleic acids encodes a repertoire ofpolypeptides. Each nucleic acid member of the library preferably has asequence related to one or more other members of the library. By relatedsequence is meant an amino acid sequence having at least 50% identity,suitably at least 60% identity, suitably at least 70% identity, suitablyat least 80% identity, suitably at least 90% identity, suitably at least95% identity, suitably at least 98% identity, suitably at least 99%identity. Identity is suitably judged across a contiguous segment of atleast 10 amino acids, suitably least 12 amino acids, suitably least 14amino acids, suitably least 16 amino acids, suitably least 17 aminoacids or the full length of the reference sequence.

As used herein, altering the reactive groups or scaffolds refers tochanging the chemical structure or composition of, or replacing, any oneor more of the reactive groups, or any part of the scaffold. It alsorefers to replacing the entire scaffold.

In one embodiment, altering the reactive groups or scaffolds will resultin a conformational change in the assembled peptide ligand. Forinstance, it includes changes which will alter bond geometries, thestructure and therefore the spatial arrangement of the reactive groupson the scaffold, the structure and therefore the spatial arrangement ofreactive groups on the polypeptide, the structure of the backbone itselfof the scaffold, and combinations of such features. Also included arechanges which affect the flexibility of bonds and linkages, which canalter the level of adaptive fit which can be expected of a peptideligand.

Conformational diversity, as referred to herein, is the degree ofdifferent conformational options for a peptide ligand in a repertoire ofpeptide ligands. If a repertoire of ligands is constructed with onescaffold, the diversity will result from variations in the peptidesequence. In accordance with the present invention, further diversitycan be achieved by varying the way in which the peptide attaches to thescaffold, or the scaffold itself. In the context of a repertoire,variation can include a number of options for any given change, thusintroducing still further diversity. The resulting repertoires thuscover a diversity space not covered by the original repertoire. Thisspace may be larger, smaller or partly overlapping.

Increasing Ligand Diversity

In general, peptide ligand repertoires may be prepared by techniquesknown in the prior art, or described herein. The basic components of theligands, especially the molecular scaffold and the polypeptidecomponents, are known from Timmerman et al., 2005 Chem Bio Chem6:821-824, as well as WO2004/077062, WO2006/078161 and WO2008/013454.The use of phage display to select polypeptides complexed with molecularscaffolds is described in Heinis et al., 2009, Nature Chemical Biology,5:502-507, as well as our copending unpublished international patentapplication PCT/GB09/000301. Each of these documents is incorporatedherein by reference. Preferred methods for constructing ligands andligand repertoires according to the invention, and the use of phagedisplay, are described in more detail below.

In general, modification of the scaffold-peptide interface can beexploited to increase repertoire diversity, or to reduce it if desired.As set forth above, there are three fundamental routes to achieving thisaim: (a) altering the reactive groups of the polypeptide; (b) alteringthe scaffold, including the reactive groups of the scaffold; (c)altering the bond between the polypeptide and the scaffold, especiallyafter the bond has been formed. Combinations of more than one method arealso possible.

(A) Construction Ligand Repertoires

(i) Molecular Scaffold

The molecular scaffold is sometimes referred to as the ‘molecular core’or ‘connector compound’. This molecule, which is in some embodiments anaromatic small molecule, comprises scaffold reactive groups which arecapable of forming covalent bonds with a peptide. Reactive groups on thepeptide interact with the scaffold reactive groups to form said covalentbonds.

Suitably the molecular scaffold may be, or may be based on, naturalmonomers such as nucleosides, sugars, or steroids. Suitably themolecular scaffold may comprise a short polymer of such entities, suchas a dimer or a trimer.

A very wide variety of scaffold structures is known, and has beencatalogued in the art, for instance in the CAS registry. See Lipkus etal., (2008) J Org Chem 73:4443-4451. Factors which contribute todiversity of structure are also known, such as the size and position ofheteroatoms.

Suitably the molecular scaffold is a compound of known toxicity,suitably of low toxicity. Examples of suitable compounds includecholesterols, nucleotides, steroids, or existing drugs such astamazepam.

Suitably the molecular scaffold may be a macromolecule. Suitably themolecular scaffold is a macromolecule composed of amino acids,nucleotides or carbohydrates.

Suitably the molecular scaffold comprises reactive groups that arecapable of reacting with functional group(s) of the target polypeptideto form covalent bonds.

The molecular scaffold of the invention contains chemical groups thatallow functional groups of the polypeptide of the encoded library of theinvention to form covalent links with the molecular scaffold. Saidchemical groups are selected from a wide range of functionalitiesincluding benzylic halides, α-halocarboxylic acids or amides, acryloylmoieties, amines, thiols, alcohols, ketones, aldehydes, nitriles,carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides,maleimides, azides, alkyl halides and acyl halides.

Suitably the molecular scaffold may comprise or may consist oftris(bromomethyl)benzene, especially 1,3,5-Tris(bromomethyl)benzene(‘TBMB’), or a derivative thereof.

In some embodiments the molecular scaffold may have a tetrahedralgeometry such that reaction of four functional groups of the encodedpolypeptide with the molecular scaffold generates not more than twoproduct isomers. Other geometries are also possible; indeed, an almostinfinite number of scaffold geometries is possible, leading to greaterpossibilities for peptide ligand diversification.

A suitable molecular scaffold is 2,4,6-Tris(bromomethyl)mesitylene. Itis similar to 1,3,5-Tris(bromomethyl)benzene but contains additionallythree methyl groups attached to the benzene ring. In the case of thisscaffold, the additional methyl groups may form further contacts withthe polypeptide and hence add additional structural constraint. Thus, adifferent diversity range is achieved than with1,3,5-Tris(bromomethyl)benzene.

Of course, the methyl groups need not be added at every position; andthe positions of the substitutions need not be symmetrical. Even furtherdiversity can, therefore, be achieved.

The molecular scaffold of the present invention is selected from eithera small molecule or a macromolecular structure. The said molecularscaffold is composed of organic, inorganic or organic and inorganiccomponents.

In a preferred embodiment, the molecular scaffold is a small organicmolecule as for example a linear alkane. More suitably the molecularscaffold is a branched alkane, a cyclic alkane, a polycyclic alkane, anaromate, a heterocyclic alkane or a heterocyclic aromate, which offerthe advantage of being less flexible (i.e. more rigid). Most suitablythe molecular scaffold comprises at least one benzylic group.

In another embodiment, the molecular scaffold is selected from amacromolecular structure as for example a polypeptide, a polynucleotideor a polysaccharide.

(ii) Polypeptide

As set forth in more detail below, the polypeptide element of thepeptide ligand comprises two or more reactive groups, preferably threeor more reactive groups, which are responsible for binding to thescaffold; and loop sequences between the reactive groups. Diversity canbe obtained, as in the prior art, by varying the sequence of the loops.The reactive group for conjugating the peptide to the molecular scaffoldis, in one embodiment, a functionalised —SH group. A preferred aminoacid having such a group is cysteine. However, other reactive groups maybe used in conjunction with or instead of cysteine in any givenpolypeptide. For example, the two reactive groups may comprise onecysteine and one further suitable reactive group, which might forexample comprise methionine, lysine, selenocysteine or other(s). Inaccordance with the present invention, further diversification can beachieved by variation of the reactive groups.

The reactive groups of the polypeptides are suitably provided by sidechains of natural or non-natural amino acids. The reactive groups of theencoded polypeptides are suitably selected from thiol groups, aminogroups, carboxyl groups, guanidinyl groups, phenolic groups or hydroxylgroups. The reactive groups of the encoded polypeptides may suitably beselected from azide, keto-carbonyl, alkyne, vinyl, or aryl halidegroups. The reactive groups of the encoded polypeptides for linking to amolecular scaffold may suitably be the amino or carboxy termini of thepolypeptide.

In some embodiments each of the reactive groups of the polypeptide forlinking to a molecular scaffold are of the same type. For example, eachreactive group may be a cysteine residue.

In some embodiments the reactive groups for linking to a molecularscaffold may comprise two or more different types, or may comprise threeor more different types. For example, the reactive groups may comprisetwo cysteine residues and one methionine or lysine residue, or maycomprise one cysteine residue, one methionine or lysine residue and oneN-terminal amine, or any group selected from the list set forth above.

Cysteine is useful in the context of peptide ligands because it has theadvantage that its reactivity is most different from all other aminoacids. Scaffold reactive groups that could be used on the molecularscaffold to react with thiol groups of cysteines are benzylic halides,.α-halocarboxylic acids or amides, acryloyl moieties, aziridines,vinylsulfones, Examples are bromomethylbenzene (the scaffold reactivegroup exemplified by TBMB). Other scaffold reactive groups that are usedto couple selectively compounds to cysteines in proteins are maleimides.Examples of maleimides which may be used as molecular scaffolds in theinvention include: tris-(2-maleimidoethyl)amine,tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. Any variationand combination of different scaffold reactive groups is also possible.

Selenocysteine is a natural amino acid which has a similar reactivity tocysteine and can be used for the same reactions. Thus, wherever cysteineis mentioned, it is typically acceptable to substitute selenocysteineunless the context suggests otherwise.

Lysines (and primary amines of the N-terminus of peptides) are alsosuited as reactive groups to modify peptides on phage by linking to amolecular scaffold. However, in the event that phage is used forselection, lysines are more abundant in phage proteins than cysteinesand there is a higher risk that phage particles might becomecross-linked or that they might lose their infectivity. Nevertheless, ithas been found that lysines are especially useful in intramolecularreactions (e.g. when a molecular scaffold is already linked to the phagepeptide) to form a second or consecutive linkage with the molecularscaffold. In this case the molecular scaffold may react preferentiallywith lysines of the displayed peptide (in particular lysines that are inclose proximity). Scaffold reactive groups that react selectively withprimary amines are activated esters such as succinimides, aldehydes oralkyl halides.

In the bromomethyl group that is used in a number of the accompanyingexamples, the electrons of the benzene ring can stabilize the cationictransition state. This particular alkyl halide is therefore 100-1000times more reactive than alkyl halides that are not connected to abenzene (more generally, an (heterocyclic) aromatic system) group.

Examples of succinimides for use as molecular scaffold includetris-(succinimidyl aminotriacetate), 1,3,5-Benzenetriacetic acid.Examples of aldehydes for use as molecular scaffold includeTriformylmethane, and benzene 1,3,5 triscarbaldehyde(1,3,5-Trisformylbenzene). Examples of alkyl halides for use asmolecular scaffold include1,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene,1,3,5-Tris(bromomethyl)benzene,1,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.

In some embodiments, molecular linkers or modifications may be added to(or to create) reactive groups of the encoded polypeptides beforeattachment of the molecular scaffold wherein said linkers ormodifications are capable to react with the molecular scaffold.

The amino acids with reactive groups for linking to a molecular scaffoldmay be located at any suitable positions within the encoded polypeptide.In order to influence the particular structures or loops created, thepositions of the amino acids having the reactive groups may be varied bythe skilled operator, e.g. by manipulation of the nucleic acid encodingthe polypeptide in order to mutate the polypeptide produced.

Each of the amino acids of the encoded polypeptide may be a target formutagenesis (e.g. restricted variance mutagenesis) according to theneeds of the skilled worker or the purpose to which the invention isbeing applied. Clearly at least two reactive groups for bonding to themolecular scaffold are required on the polypeptide of interest. Aminoacids other than those required for bonding to the molecular scaffoldmay be freely varied according to operator needs and are termed‘variable amino acids’. Said variable amino acids of the encodedpolypeptide (e.g. polypeptide library member(s)) may be randomised,partially randomised, or constant.

The polypeptide comprises a molecular scaffold binding segment. This isthe region to which the molecular scaffold is attached. Suitably thecommentary regarding reactive groups on the polypeptide is applied tothis binding segment. Suitably the molecular scaffold binding segment ofthe target polypeptide comprises 1 to 27 amino acid residues, suitably 5to 20 amino acid residues. Suitably the molecular scaffold bindingsegment of the target polypeptide comprises fewer than 10 amino acids.This has the advantage of imposing further conformational constraintonto the polypeptide segment when it is attached to the molecularscaffold.

The target polypeptide suitably comprises the sequence XC(X)₆C(X)₆CX,wherein X stands for a random natural amino acid. Suitably, in theformulae mentioned herein, C may be replaced with another amino acidwhich forms a covalent linkage with a scaffold reactive group.

The target polypeptide suitably comprises the sequence(X)_(l)Y(X)_(m)Y(X)_(n)Y(X)_(o), wherein Y represents an amino acid witha reactive group, X represents a random amino acid, m and n are numbersbetween 1 and 20 defining the length of intervening polypeptide segmentsand l and o are numbers between 0 and 20 defining the length of theflanking polypeptide segments.

In some embodiments, the peptide ligand of the invention may comprise apolypeptide with the sequence C(X)₆C(X)₆C. In one embodiment, a librarymember or peptide ligand of the invention may comprise a mesitylenemolecular scaffold and a polypeptide with the sequence C(X)₆C(X)₆C,wherein the polypeptide is tethered to the exocyclic methyl groups ofthe molecular scaffold via the cysteine residues of the polypeptideforming three thioether bonds therewith, and wherein X stands for anamino acid, (suitably a natural amino acid).

In one embodiment of the invention, at least one of the reactive groupsof the polypeptides is orthogonal to the remaining reactive groups. Theuse of orthogonal reactive groups allows the directing of saidorthogonal reactive groups to specific sites of the molecular core.Linking strategies involving orthogonal reactive groups may be used tolimit the number of product isomers formed. In other words, by choosingdistinct or different reactive groups for one or more of the at leastthree bonds to those chosen for the remainder of the at least threebonds, a particular order of bonding or directing of specific reactivegroups of the polypeptide to specific positions on the molecularscaffold may be usefully achieved.

In another embodiment, the reactive groups of the encoded polypeptide ofthe invention are reacted with molecular linkers wherein said linkersare capable to react with a molecular scaffold so that the linker willintervene between the molecular scaffold and the polypeptide in thefinal bonded state.

Suitable amino acids of the members of the genetically encodedcombinatorial chemical libraries can be replaced by any natural ornon-natural amino acid. Excluded from these exchangeable amino acids arethe ones harbouring functional groups for cross-linking the polypeptidesto a molecular core. A group of adjacent amino acids that can be variedis defined as a polypeptide segment. The size of a single polypeptidesegment suitably ranges from 1 to 20 amino acids. The polypeptidesegments have either random sequences, constant sequences or sequenceswith random and constant amino acids. The amino acids with reactivegroups are either located in defined or random positions within theencoded polypeptide of the invention.

In one embodiment, the polypeptide segments that are bounded by twoamino acids harbouring reactive groups for bonding with a molecularscaffold/molecular core are short amino acid sequences of 10 or feweramino acids. Reaction of said encoded polypeptide sequences with amolecular core generates library members with high conformationalconstraint. Conformational constrained ligands are generally morespecific and have higher binding affinities. The conformationalconstraint can also protect the ligands from proteolytic degradation forexample in bodily fluids.

In one embodiment, an encoded polypeptide with three reactive groups hasthe sequence (X)_(l)Y(X)_(m)Y(X)_(n)Y(X)_(o) (SEQ ID No. 1), wherein Yrepresents an amino acid with a reactive group, X represents a randomamino acid, m and n are numbers between 1 and 20 defining the length ofintervening polypeptide segments and l and o are numbers between 0 and20 defining the length of the flanking polypeptide segments.

In a preferred embodiment, an encoded polypeptide library of theinvention has the sequence AC(X)₆C(X)₆CG, wherein A represents alanine,C represents cysteine, X represents a random natural amino acid and Grepresents glycine.

Alternatives to thiol-mediated conjugations can be used to attach themolecular scaffold to the peptide via covalent interactions.Alternatively these techniques may be used in modification or attachmentof further moieties (such as small molecules of interest which aredistinct from the molecular scaffold) to the polypeptide after they havebeen selected or isolated according to the present invention—in thisembodiment then clearly the attachment need not be covalent and mayembrace non-covalent attachment. These methods may be used instead of(or in combination with) the thiol mediated methods by producing phagethat display proteins and peptides bearing unnatural amino acids withthe requisite chemical reactive groups, in combination small moleculesthat bear the complementary reactive group, or by incorporating theunnatural amino acids into a chemically or recombinantly synthesisedpolypeptide when the molecule is being made after theselection/isolation phase.

The unnatural amino acids incorporated into peptides and proteins onphage may include 1) a ketone reactive group (as found in para or metaacetyl-phenylalanine) that can be specifically reacted with hydrazines,hydroxylamines and their derivatives (Addition of the keto reactivegroup to the genetic code of Escherichia coli. Wang L, Zhang Z, Brock A,Schultz P G. Proc Natl Acad Sci USA. 2003 Jan. 7; 100(1):56-61; BioorgMed Chem Lett. 2006 Oct. 15; 16(20):5356-9. Genetic introduction of adiketone-containing amino acid into proteins. Zeng H, Xie J, Schultz PG), 2) azides (as found in p-azido-phenylalanine) that can be reactedwith alkynes via copper catalysed “click chemistry” or strain promoted(3+2) cyloadditions to form the corresponding triazoles (Addition ofp-azido-L-phenylalanine to the genetic code of Escherichia coli. Chin JW, Santoro S W, Martin A B, King D S, Wang L, Schultz P G. J Am ChemSoc. 2002 Aug. 7; 124(31):9026-7; Adding amino acids with novelreactivity to the genetic code of Saccharomyces cerevisiae. Deiters A,Cropp T A, Mukherji M, Chin J W, Anderson J C, Schultz P G. J Am ChemSoc. 2003 Oct. 1; 125(39):11782-3), or azides that can be reacted witharyl phosphines, via a Staudinger ligation (Selective Staudingermodification of proteins containing p-azidophenylalanine. Tsao M L, TianF, Schultz P G. Chem bio chem. 2005 December; 6(12):2147-9), to form thecorresponding amides, 4) Alkynes that can be reacted with azides to formthe corresponding triazole (In vivo incorporation of an alkyne intoproteins in Escherichia coli. Deiters A, Schultz P G. Bioorg Med ChemLett. 2005 Mar. 1; 15(5):1521-4), 5) Boronic acids (boronates) than canbe specifically reacted with compounds containing more than oneappropriately spaced hydroxyl group or undergo palladium mediatedcoupling with halogenated compounds (Angew Chem Int Ed Engl. 2008;47(43):8220-3. A genetically encoded boronate-containing amino acid,Brustad E, Bushey M L, Lee J W, Groff D, Liu W, Schultz P G), 6) Metalchelating amino acids, including those bearing bipyridyls, that canspecifically co-ordinate a metal ion (Angew Chem Int Ed Engl. 2007;46(48):9239-42. A genetically encoded bidentate, metal-binding aminoacid. Xie J, Liu W, Schultz P G).

Unnatural amino acids may be incorporated into proteins and peptidesdisplayed on phage by transforming E. coli with plasmids or combinationsof plasmids bearing: 1) the orthogonal aminoacyl-tRNA synthetase andtRNA that direct the incorporation of the unnatural amino acid inresponse to a codon, 2) The phage DNA or phagemid plasmid altered tocontain the selected codon at the site of unnatural amino acidincorporation (Proc Natl Acad Sci USA. 2008 Nov. 18; 105(46):17688-93.Protein evolution with an expanded genetic code. Liu C C, Mack A V, TsaoM L, Mills J H, Lee H S, Choe H, Farzan M, Schultz P G, Smider V V; Aphage display system with unnatural amino acids. Tian F, Tsao M L,Schultz P G. J Am Chem Soc. 2004 Dec. 15; 126(49):15962-3). Theorthogonal aminoacyl-tRNA synthetase and tRNA may be derived from theMethancoccus janaschii tyrosyl pair or a synthetase (Addition of aphotocrosslinking amino acid to the genetic code of Escherichia coli.Chin J W, Martin A B, King D S, Wang L, Schultz P G. Proc Natl Acad SciUSA. 2002 Aug. 20; 99(17):11020-4) and tRNA pair that naturallyincorporates pyrrolysine (Multistep engineering of pyrrolysyl-tRNAsynthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl)lysine for site-specific protein modification. Yanagisawa T, Ishii R,Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Chem Biol. 2008 Nov.24; 15(11):1187-97; Genetically encoding N(epsilon)-acetyllysine inrecombinant proteins. Neumann H, Peak-Chew S Y, Chin J W. Nat Chem Biol.2008 April; 4(4):232-4. Epub 2008 Feb. 17). The codon for incorporationmay be the amber codon (UAG) another stop codon (UGA, or UAA),alternatively it may be a four base codon. The aminoacyl-tRNA synthetaseand tRNA may be produced from existing vectors, including the pBK seriesof vectors, pSUP (Efficient incorporation of unnatural amino acids intoproteins in Escherichia coli. Ryu Y, Schultz P G. Nat Methods. 2006April; 3(4):263-5) vectors and pDULE vectors (Nat Methods. 2005 May;2(5):377-84. Photo-cross-linking interacting proteins with a geneticallyencoded benzophenone. Farrell I S, Toroney R, Hazen J L, Mehl R A, ChinJ W). The E. coli strain used will express the F′ pilus (generally via atra operon). When amber suppression is used the E. coli strain will notitself contain an active amber suppressor tRNA gene. The amino acid willbe added to the growth media, preferably at a final concentration of 1mM or greater. Efficiency of amino acid incorporation may be enhanced byusing an expression construct with an orthogonal ribosome binding siteand translating the gene with ribo-X (Evolved orthogonal ribosomesenhance the efficiency of synthetic genetic code expansion. Wang K,Neumann H, Peak-Chew S Y, Chin J W. Nat Biotechnol. 2007 July;25(7):770-7). This may allow efficient multi-site incorporation of theunnatural amino acid providing multiple sites of attachment to theligand.

(iv) Post Attachment Modification

In some embodiments the polypeptide-molecular scaffold complex may bemodified at a time following attachment.

Protease Cleavage

In some embodiments, the polypeptide elements of the invention areproteolytically cleaved once they are tethered to a molecularscaffold/molecular core. The cleavage generates ligands having discretepeptide fragments tethered to a molecular scaffold/molecular core.

For example, one or more amide bonds of the polypeptide may beproteolytically cleaved after tethering the polypeptide to the molecularcore. This has the advantage of creating short polypeptides, each joinedto the molecular scaffold by at least one covalent bond, but whichpresent different molecular structures which are retained in a complexcomprising the nucleic acid encoding the parent polypeptide. Thepolypeptide cleavage is suitably catalysed by any suitable means knownin the art such as controlled hydrolysis or more suitably enzymaticcleavage by a suitable protease. The protease may be any suitableprotease but is preferably a protease with a specific polypeptiderecognition sequence or motif. This advantageously leads to productionof more defined and/or more predictable polypeptide cleavage products.Indeed, in this embodiment, protease recognition sequences may besystematically added or removed from the target polypeptide, for exampleby manipulation of the nucleic acid(s) encoding it. This advantageouslyprovides a greater degree of control and permits greater diversity to beproduced in the molecules displayed according to the present invention.Thus, enhanced diversity may be obtained by varying the nature andposition of protease cleavage sites in a polypeptide molecule. Mostsuitably the polypeptide comprises at least one protease recognitionsite. Suitably each said cleavage site is comprised within amino acidsequence(s) in between reactive groups on the polypeptide used forcovalent bonding to the molecular scaffold. Suitably each saidrecognition site is comprised within amino acid sequence(s) in betweenreactive groups on the polypeptide used for covalent bonding to themolecular scaffold.

The peptide loops are suitably cleaved with a protease that recognizesand processes polypeptides at specific amino acid positions such astrypsin (arginine or lysine in P1 position) or thermolysin (aliphaticside chains in P1 position). The enzyme is used at a concentration thatallows efficient processing of the peptide loops of the displayedmolecule but spares the phage particle. The optimal conditions can varydepending on the length of the polypeptide loops and on the proteaseused. Trypsin for example is typically used at 200 nM in TBS-Ca buffer(25 mM Tris HCl/137 mM NaCl/1 mM CaCl₂, pH 7.4) for 10 min at 10° C. Awhole range of proteases that are suitable to modify displayedpolypeptides but that spare the phage are described in Kristensen, P.and Winter, G. (Proteolytic selection for protein folding usingfilamentous bacteriophages; Fold Des. 1998; 3(5):321-8). The enzymaticprocessing of peptide on phage may be a ‘partial proteolysis’ since itcannot be excluded that a limited number of phage coat proteins arecleaved. Thus in optimisation of the conditions, the best balancebetween maximised cleavage of the target and maximum sparing of thephage particles is suitably chosen.

Suitably the target polypeptide comprises at least one such proteolyticcleavage site. Suitably the target polypeptide comprises at least twosuch proteolytic cleavage sites.

Suitably the target polypeptide comprises at least three suchproteolytic cleavage sites.

In each such proteolysis embodiment, suitably the said protease site(s)are located within the target polypeptide loops subtended by themolecular scaffold. This has the advantage that the molecular scaffoldis retained on the complex, as otherwise the polypeptide-molecularscaffold complex may be separated from the nucleic acid encoding thetarget polypeptide, which is undesirable for the majority ofapplications of the invention.

The use of short loops (short being e.g. 6 amino acid residues or less)may compromise the ability of some proteases to cleave within the loops.In this case it may be desirable to select longer loops which are likelyto be more accessible to the protease. Furthermore after cleavage of theloops by endoprotease, it may be desirable to cut back the loops furtherwith other endoproteases, or indeed by exoproteases, such ascarboxypeptidases or aminopeptidases.

When the target polypeptide comprises more than one such protease site,suitably each of the sites occurs between two covalent bonds madebetween the target polypeptide and the molecular scaffold. Multiplecleavage sites may occur between bonds if necessary.

In cleavage embodiments, suitably the parent polypeptide will beconsidered as a whole for the assessment of whether or not it isattached to the molecular scaffold by at least two or three covalentbonds. More suitably the target polypeptide will be considered to be theintact (uncleaved) polypeptide when assessing whether or not it isattached to the molecular scaffold by at least three covalent bonds.

Protease Resistance

In another embodiment, the polypeptides may be resistant to proteasecleavage. In general, tightly folded polypeptide structures are moreresistant to proteases, since the protease cannot physically access thepolypeptide to cleave it. Therefore, manipulation of the scaffold andscaffold attachment in the peptide ligand can modulate proteasesensitivity, by influencing the folding of the polypeptide loop.

As indicated in the preceding section, a protease step can be introducedto cleave accessible sites within loops attached to a chemical scaffold.If a repertoire of peptide conjugates is displayed on phage, this leadsto peptides each joined to the chemical scaffold by at least onecovalent bond, but retained in a complex comprising the nucleic acidencoding the parent polypeptide. The treatment of the chemicallymodified phage with protease before selection with antigen is expectedto give rise to phage bearing peptide conjugates with cleaved loop(s),and also to phage bearing peptide conjugates with uncleaved loop(s) dueto lack of a cleavage site, or otherwise being resistant to cleavage. Itis possible to distinguish these species if one binds to antigen and theother does not, by comparing the binding of the selected phage clones totarget antigen before and after protease treatment. Thus the specieswith cleaved loops will be expected to bind after protease treatment,but not before; whereas the protease-resistant species will be expectedto bind both before and after treatment. Note that if a conjugate bindswith both cleaved and uncleaved loops (as with PK15 after kallikreincleavage; see Heinis et al, 2009), it may be incorrectly identified asprotease resistant. This shows the importance of using a direct methodfor checking cleavage, for example by synthesizing the peptideconjugates chemically, and checking for evidence of cleavage, forexample by mass spectrometry.

If cleaved loop conjugates are preferred to protease resistantconjugates, it will be advantageous to treat the chemically modifiedphage repertoire with protease before the first round of selection, andto continue to use the same protease, or one with a common cut-site, insubsequent rounds. However protease resistant conjugates mayalternatively be desired. Such peptides may be useful for oraladministration to survive the gut proteases, or those otherwise subjectto proteolytic attack in the blood, tissues or cells. In this case, afirst round of selection without protease, followed by a subsequentround of selection with protease, should favour the selection of theresistant species.

The use of protease has further utility during the selection process.For example, some unformed loops (linear segments of sequence) may bepresent in the libraries because (a) errors in the synthesis of thenucleotides have failed to encode a required cysteine residue, or (b) arequired cysteine residue has made a disulphide bond to free cysteine insolution (perhaps due to inadequate reduction or re-oxidation), or hasreacted in an irreversible manner (for example is oxidized to cysteicacid, or one of the required cysteines has reacted with a differentmolecule of the scaffold to the others). As linear segments of sequenceare more susceptible to protease attack than loops, then, subject to acleavage site being present, it may be possible to avoid such bindersusing protease.

A protease step (in the presence of reducing agent) is also advantageousto eliminate loops that have formed via disulphides between the requiredcysteines rather than through the chemical scaffold. This may beexpected if there is inadequate reduction (or subsequent reoxidation) ofthe cysteines on the phages. For this reason we used degassed buffersduring the chemical cross-linking step; we also kept low levels of thereducing agent (TCEP) during the reaction with TBMB to maintain thereducing environment. Nevertheless, after the first round of selection,we found many sequences that included four cysteine residues (the threerequired cysteine residues, and a further cysteine residue in the loop),for example CFNSEWSCLQSCSNC (SEQ ID No.2) in the selections ofrepertoires against MDM2 (see Example 1). Such extra cysteines areexpected to be present in the peptide repertoires, as the syntheticnucleotide library includes random codons (NNK diversity: where Nrepresents a 25% mix each of adenine, thymine, guanine, and cytosinenucleotides, and K represents a 50% mix each of thymine and guaninenucleotides). Under some conditions, for example if there is inadequatereduction, or incomplete reaction of the required cysteines with thechemical scaffold (perhaps due to competing reactions for the scaffoldby amino groups or water), an extra cysteine may be expected, underoxidising conditions, to form disulphide loops with one of the threerequired cysteines. Alternatively an extra cysteine may react with thescaffold, leaving two of the required cysteines to formdisulphide-closed loops.

Whatever the exact mechanism behind their generation, suchdisulphide-closed loops may compete with the scaffold-closed loops, andpredominate. It should be possible to reduce the frequency of the extracysteines by using synthetic nucleotide libraries built from triplets,rather than monomers, so avoiding cysteine codons in the loops; and/orto undertake the selections in the presence of reducing agent, so as toopen the disulphide-closed loops. More conveniently we have found thatthe treatment of the chemically modified phage repertoires with proteasein the presence of reducing agent (such as dithiothreitol), so as toopen and then cleave the loops, helps to minimise the contribution ofsuch species.

In a preferred embodiment, therefore, the polypeptide ligands of theinvention are substantially protease resistant. The invention thereforeprovides a method for selecting a peptide ligand having increasedprotease resistance, comprising the steps of:

(a) providing a first repertoire of polypeptides;(b) conjugating said polypeptides to a molecular scaffold which binds tothe polypeptides at two or more amino acid residues, to form arepertoire of polypeptide conjugates;(c) screening said repertoire for binding against a target, andselecting members of the first repertoire which bind to the target;(d) optionally treating the selected repertoire with reducing agent(d) subjecting the repertoire to selection for protease resistance; and(e) further screening said repertoire for binding to the target.

In the most preferred embodiment, the protease step is included beforethe screening of repertoire. The invention accordingly provides a methodof selecting a peptide ligand having increased protease resistance,comprising the steps of:

(a) providing a first repertoire of polypeptides;(b) conjugating said polypeptides to a molecular scaffold which binds tothe polypeptides at two or more amino acid residues, to form arepertoire of polypeptide conjugates;(c) optionally treating the repertoire with reducing agent(d) subjecting the repertoire to selection for protease resistance; and(e) screening said repertoire for binding against a target, andselecting members of the first repertoire which bind to the target.

A screen for protease resistance can simply take the form of limiteddigestion with a protease; those members of the repertoire which aresensitive to proteases will be eliminated. Most desirable will be to usea protease that is active under the conditions in which the bicyclicpeptide will be used, for example in the presence of serum.

Bond Modification

Bonds in the scaffold, or between the scaffold and the polypeptide, maybe modified post-attachment to change the structure of the peptideligand. For example, thioether bonds may oxidized to sulphones, whichalters the stereochemistry of the bond and therefore the structure ofthe peptide loop(s) subtended at the scaffold attachment point.

More generally, groups containing heteroatoms may be oxidized or reducedto alter bond stereochemistry, thus introducing further levels ofdiversity into the peptide ligand.

(v) Synthesis

It should be noted that once the polypeptide of interest is isolated oridentified according to the present invention, then its subsequentsynthesis may be simplified wherever possible. For example, the sequenceof the polypeptide of interest may be determined, and it may bemanufactured synthetically by standard techniques followed by reactionwith a molecular scaffold in vitro. When this is performed, standardchemistry may be used since there is no longer any need to preserve thefunctionality or integrity of the genetically encoded carrier particle.This enables the rapid large scale preparation of soluble material forfurther downstream experiments or validation. In this regard, largescale preparation of the candidates or leads identified by the methodsof the present invention could be accomplished using conventionalchemistry such as that disclosed in Timmerman et al.

Thus, the invention also relates to manufacture of polypeptides orconjugates selected as set out herein, wherein the manufacture comprisesoptional further steps as explained below. Most suitably these steps arecarried out on the end product polypeptide/conjugate made by chemicalsynthesis, rather than on the phage.

Optionally amino acid residues in the polypeptide of interest may besubstituted when manufacturing a conjugate or complex e.g. after theinitial isolation/identification step.

Peptides can also be extended, to incorporate for example another loopand therefore introduce multiple specificities. To extend the peptide,it may simply be extended chemically at its N-terminus or C-terminususing standard solid phase or solution phase chemistry. Standardactivation/bioconjugation chemistry may be used to introduce anactivatable N- or C-terminus. Alternatively additions may be made byfragment condensation or native chemical ligation e.g. as described in(Dawson P E, Muir T W, Clark-Lewis I, Kent, S B H. 1994. Synthesis ofProteins by Native Chemical Ligation. Science 266:776-779), or byenzymes, for example using subtiligase as described in (Subtiligase: atool for semisynthesis of proteins Chang T K, Jackson D Y, Burnier J P,Wells J A Proc Natl Acad Sci USA. 1994 Dec. 20; 91(26):12544-8 or inBioorganic & Medicinal Chemistry Letters Tags for labelling proteinN-termini with subtiligase for proteomics Volume 18, Issue 22, 15 Nov.2008, Pages 6000-6003 Tags for labeling protein N-termini withsubtiligase for proteomics Hikari A. I. Yoshihara, Sami Mahrus and JamesA. Wells).

Alternatively, the peptides may be extended or modified by furtherconjugation through disulphide bonds. This has the additional advantageof allowing first and second peptide to dissociate from each other oncewithin the reducing environment of the cell. In this case, the molecularscaffold (eg. TBMB) could be added during the chemical synthesis of thefirst peptide so as to react with the three cysteine groups; a furthercysteine could then be appended to the N-terminus of the first peptide,so that this cysteine only reacted with a free cysteine of the secondpeptide.

Similar techniques apply equally to the synthesis/coupling of twobicyclic macrocycles. Furthermore, addition of other functional groupsor effector groups may be accomplished in the same manner, usingappropriate chemistry, coupling at the N- or C-termini or via sidechains. Suitably the coupling is conducted in such a manner that it doesnot block the activity of either entity.

In one aspect, the molecular scaffold may be replaced in the synthesisedmolecule with another scaffold which is structurally equivalent. In suchan embodiment, the structure of the peptide ligand, and therefore itsspecificity, preferably does not change.

The advantage of this approach is that alternative chemistries can beintroduced which may be advantageous in a clinical or other setting, ormay be less costly, but cannot be contemplated in the context ofbiological selection. For example, when using phage, chemicalmanipulation is subject to constraints as it may affect phageinfectivity. Such constraints may be removed when the peptide ligand issynthesised, since phage infectivity is no longer an issue.

(B) Repertoires of Peptide Ligands

(i) Construction of Libraries

Libraries intended for selection may be constructed using techniquesknown in the art, for example as set forth in WO2004/077062, orbiological systems, including phage vector systems as described herein.Other vector systems are known in the art, and include other phage (forinstance, phage lambda), bacterial plasmid expression vectors,eukaryotic cell-based expression vectors, including yeast vectors, andthe like.

Non-biological systems such as those set forth in WO2004/077062 arebased on conventional chemical screening approaches. They are simple,but lack the power of biological systems since it is impossible, or atleast impracticably onerous, to screen large libraries of peptideligands. However, they are useful where, for instance, only a smallnumber of peptide ligands needs to be screened. Screening by suchindividual assays, however, may be time-consuming and the number ofunique molecules that can be tested for binding to a specific targetgenerally does not exceed 10⁶ chemical entities.

In contrast, biological screening or selection methods generally allowthe sampling of a much larger number of different molecules. Thusbiological methods are more suitably used in application of theinvention. In biological procedures, molecules are assayed in a singlereaction vessel and the ones with favourable properties (i.e. binding)are physically separated from inactive molecules. Selection strategiesare available that allow to generate and assay simultaneously more than10¹³ individual compounds. Examples for powerful affinity selectiontechniques are phage display, ribosome display, mRNA display, yeastdisplay, bacterial display or RNA/DNA aptamer methods. These biologicalin vitro selection methods have in common that ligand repertoires areencoded by DNA or RNA. They allow the propagation and the identificationof selected ligands by sequencing. Phage display technology has forexample been used for the isolation of antibodies with very high bindingaffinities to virtually any target.

When using a biological system, once a vector system is chosen and oneor more nucleic acid sequences encoding polypeptides of interest arecloned into the library vector, one may generate diversity within thecloned molecules by undertaking mutagenesis prior to expression;alternatively, the encoded proteins may be expressed and selected beforemutagenesis and additional rounds of selection are performed.

Such approaches are particularly indicated for affinity maturation ofpeptide ligands as described herein. Foe example, a first and a secondrepertoire of peptide ligands which bind to a first and second targetmay be combined, and the resulting third repertoire subjected toaffinity maturation by mutagenesis of the nucleic acid library memberswhich encode the repertoire.

Mutagenesis of nucleic acid sequences encoding structurally optimisedpolypeptides is carried out by standard molecular methods. Of particularuse is the polymerase chain reaction, or PCR, (Mullis and Faloona (1987)Methods Enzymol., 155: 335, herein incorporated by reference). PCR,which uses multiple cycles of DNA replication catalysed by athermostable, DNA-dependent DNA polymerase to amplify the targetsequence of interest, is well known in the art. The construction ofvarious antibody libraries has been discussed in Winter et al. (1994)Ann. Rev. Immunology 12, 433-55, and references cited therein.

PCR is performed using template DNA (at least Ifg; more usefully, 1-1000ng) and at least 25 pmol of oligonucleotide primers; it may beadvantageous to use a larger amount of primer when the primer pool isheavily heterogeneous, as each sequence is represented by only a smallfraction of the molecules of the pool, and amounts become limiting inthe later amplification cycles. A typical reaction mixture includes: 2μl of DNA, 25 pmol of oligonucleotide primer, 2.5 μl of 10×PCR buffer 1(Perkin-Elmer, Foster City, Calif.), 0.4, μl of 1.25 μM dNTP, 0.15 μl(or 2.5 units) of Taq DNA polymerase (Perkin Elmer, Foster City, Calif.)and deionized water to a total volume of 25 μl. 1. Mineral oil isoverlaid and the PCR is performed using a programmable thermal cycler.The length and temperature of each step of a PCR cycle, as well as thenumber of cycles, is adjusted in accordance to the stringencyrequirements in effect. Annealing temperature and timing are determinedboth by the efficiency with which a primer is expected to anneal to atemplate and the degree of mismatch that is to be tolerated; obviously,when nucleic acid molecules are simultaneously amplified andmutagenized, mismatch is required, at least in the first round ofsynthesis. The ability to optimise the stringency of primer annealingconditions is well within the knowledge of one of moderate skill in theart. An annealing temperature of between 30° C. and 72° C. is used.Initial denaturation of the template molecules normally occurs atbetween 92° C. and 99° C. for 4 minutes, followed by 20-40 cyclesconsisting of denaturation (94-99 C for 15 seconds to 1 minute),annealing (temperature determined as discussed above; 1-2 minutes), andextension (72° C. for 1-5 minutes, depending on the length of theamplified product). Final extension is generally for 4 minutes at 72°C., and may be followed by an indefinite (0-24 hour) step at 4° C.

Alternatively, given the short chain lengths of the polypeptidesaccording to the invention, the variants are preferably synthesised denovo and inserted into suitable expression vectors. Peptide synthesiscan be carried out by standard techniques known in the art, as describedabove. Automated peptide synthesisers are widely available, such as theApplied Biosystems ABI 433 (Applied Biosystems, Foster City, Calif.,USA)

(ii) Genetically Encoded Diversity

The polypeptides of interest are suitably genetically encoded. Thisoffers the advantage of enhanced diversity together with ease ofhandling. An example of a genetically encoded polypeptide library is amRNA display library. Another example is a replicable genetic displaypackage (rgdp) library such as a phage display library. Suitably, thepolypeptides of interest are genetically encoded as a phage displaylibrary.

Thus, suitably the complex of the invention comprises a replicablegenetic display package (rgdp) such as a phage particle. In theseembodiments, suitably the nucleic acid is comprised by the phage genome.In these embodiments, suitably the polypeptide is comprised by the phagecoat.

In some embodiments, the invention may be used to produce a geneticallyencoded combinatorial library of polypeptides which are generated bytranslating a number of nucleic acids into corresponding polypeptidesand linking molecules of said molecular scaffold to said polypeptides.

The genetically encoded combinatorial library of polypeptides may begenerated by phage display, yeast display, ribosome display, bacterialdisplay or mRNA display.

Suitably the genetically encoded combinatorial library of polypeptidesis generated by phage display. In phage display embodiments, suitablythe polypeptides are displayed on phage according to establishedtechniques such as described below. Most suitably such display isaccomplished by fusion of the target polypeptide of interest to anengineered gene permitting external display of the polypeptide ofinterest; suitably said engineered gene comprises an engineered gene 9(p9 or gene IX), gene 8 (gene VIII), gene 7 (p7 or gene VII), gene 6 (p6or gene VI) or gene 3 (p3 or gene III) of the phage. These proteinsoffer the advantage that they contain fewer or no cysteines that canreact with molecular scaffolds such as TBMB and produce side products.For p6, it is advantageous to mutate cysteine 84 to serine. Thecysteines in p7 and p9 are most likely buried and therefore may notnecessarily need to be mutated to remove them. p8 offers the advantagethat it does not contain a cysteine residue. Thus, more suitably saidengineered gene comprises an engineered gene 8 (gene VIII), gene 6 (geneVI) or gene 3 (gene III) of the phage.

Most suitably such display is accomplished by fusion of the targetpolypeptide of interest to an engineered gene 3 protein lacking cysteineresidues in domain 1 and 2. This fusion may be accomplished by anysuitable technique known in the art such as by manipulation of thenucleic acid encoding the phage gene III protein to change the codonsencoding cysteine to codon(s) encoding other amino acid(s), and byinserting a nucleic acid sequence encoding the target polypeptide intothe gene III coding sequence in frame so that it is displayed as a geneIII fusion protein on the outside of the phage particle.

It is a benefit of the invention that the resulting engineered gene(s)leave the phage infective i.e. capable of infection and propagation.Even when the engineered gene is a gene other than gene 3, (such as gene6 or gene 8), it may still be desirable to engineer gene 3 to remove oneor more of the cysteine residue(s) (such as all of the cysteineresidues).

In a preferred embodiment, the genetically encoded polypeptides of theinvention are generated by translating a nucleic acid and linking thegenerated polypeptide to said code.

The linkage of phenotype with the genotype allows propagating ordecoding the encoded ligand repertoires. Various techniques areavailable to link the polypeptide to its polynucleotide code. Thetechniques include phage display, ribosome display, mRNA display, yeastdisplay and bacterial display and others. Encoded polypeptiderepertoires comprising up to 10exp13 individual members have beengenerated with said methods. The number of individual ligands that canbe generated according to the invention outperforms clearly the numberof individual molecules that are generally assayed in conventionalscreens.

In a preferred embodiment, phage display technology is used togenetically encode polypeptides of the invention. Phage display is amethod in which the gene of a polypeptide is fused to the gene of aphage coat protein. When phage are produced in a bacterial cell, thepolypeptide is expressed as a fusion of the coat protein. Upon assemblyof a phage particle the polypeptide is displayed on the surface of thephage. By contacting a phage repertoire with an immobilized antigen somephage remain bound to the antigen while others are removed by washing.The phage can be eluted and propagated. The DNA encoding the polypeptideof selected phage can be sequenced. Phage display can be used to encodemore than 10¹⁰ individual polypeptides. A favourable aspect of phagedisplay is that the genetic code, a single stranded DNA is packed in acoat. The coat may protect the DNA from reaction with the molecularcore.

In another preferred embodiment, the polypeptide library of theinvention is displayed on phage as a gene 3 protein fusion. Each phageparticle has about 3 to 5 copies of said phage coat protein. As a resultof the display of multiple copies of the modified polypeptide, ligandswith micromolar affinities (weak binders) can also be isolated in phageselections. Alternatively, phagemids are used to reduce the number ofpolypeptides per phage to avoid avidity effects and select ligands withhigher affinities.

In another preferred embodiment, phage with modified coat proteins areused for encoding the polypeptide libraries of the invention. Inparticular, phage lacking or having a reduced number of a specific typeof amino acid in coat proteins are used. Using said coat proteins can beadvantageous when the molecular core is reactive towards said specifictype of amino acid. This is explicitly the case when the reactive groupsof the displayed polypeptide for cross-linking a molecular core arenatural amino acids and the same type of natural amino acid is presentat a surface exposed region in the phage coat. Using said phage withmodified coat proteins can prevent cross-linking of phage particlesthrough reaction of amino acids of multiple phage with the samemolecular core. In addition, using said phage can reduce thecross-linkage of both, amino acid side chains of the reactive groups inthe polypeptide and of phage coat protein to the same molecular core.

In yet another preferred embodiment, phage with a gene 3 protein lackingthe cysteine residues of the disulfide bridges C7-C36, C46-053,C188-C201 in domain 1 and 2 are used to display polypeptide libraries ofthe invention. A phage with mutations in said positions (C7C, 0361,0461, C53V, C188V, C201A) and 14 additional mutations in the gene 3protein to compensate for the reduced thermal stability (T131, N15G,R29W, N39K, G55A, T561, 160V, T101I, Q129H, N138G, L198P, F199L, S207L,D209Y) was generated by Schmidt F. X. and co-workers (Kather, 1. et al.,J. Mol. Biol., 2005). Phage without thiol groups in said minor coatprotein are suited if one or more of the functional amino acids forcross-linking the polypeptide to a molecular core are cysteine residues.Removal of the cysteine residues in the phage coat protein preventstheir interference with said bonding reaction between polypeptide andmolecular scaffold.

This exemplary phage for application in the invention is now describedin more detail.

The disulfide-free phage of FX Schmid (domains D1-D2) comprises fd phagederived from vector fCKCBS (Krebber, C., 1997, J. Mol. Biol.). Thevector fCKCBS is based on a fd phage vector that is derived from theAmerican Type Culture Collection (ATCC: 15669-B2).

The amino acid sequence of the domains 1 and 2 of p3 of the wild-type fdphage is publicly available, for example in the PubMed database. Forease of reference, an exemplary sequence is:

(SEQ ID No. 3) AETVESCLAKPHTENSFTNVWKDDKTLDRYANYEGCLWNATGVVVCTGDETQCYGTWVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYTYINPLDGTYPPGTEQNPANPNPSLEESQPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDCAFHSGFNEDPFVCEYQGQSSDLPQPPVNAPSG

FX Schmid and co-workers had evolutionarily stabilized the p3 of thisphage (Martin, A. and Schmid, F X., 2003, J. Mol. Biol.) by mutating 4amino acids. In a consecutive work FX Schmid and co-workers had mutated6 cysteines to eliminate the 3 disulfide-bridges and inserted additionalmutations to compensate for the loss of stability (Kather, I. and SchmidF X., 2005, J. Mol. Biol.). In multiple evolutionary cycles they hadgenerated clones 19, 20, 21, and 23 which have all a disulfide-free p3with varying thermal stabilities.

The mutant 21 (‘clone 21’) can be made as described, or simply obtainedfrom FX Schmid and co-workers. According to the publication of FX Schmidthis clone contains the following mutations in the domains 1 and 2: C7S,T131, N15G, R29W, 0361, N39K, 0461, C53V, G55A, T101I, Q129H, C188V,F199L, C201A, D209Y. In addition we found the following mutations in thedomains 1 and 2 when we sequenced the clone and compared it to wild-typefd phage: P11 S and P198L. Without wishing to be bound by theory it isassumed that these mutations were already present in the phage of vectorfCKCBS.

The domains D1 and D2 of clone 21 have the following amino acidsequence:

(SEQ ID No. 4) AETVESSLAKSHIEGSFINVWKDDKILDWYANYEGILWKATGVVVITGDETQVYATVVVPIGLAIPENEGGGSEGGGSEGGGSEGGGTKPPEYGDTPIPGYIYINPLDGTYPPGTEQNPANPNPSLEESHPLNTFMFQNNRFRNRQGALTVYTGTVTQGTDPVKTYYQYTPVSSKAMYDAYWNGKFRDVAFHSGFNEDLLVAEYQGQSSYLPQPPVNAPSG

In one embodiment, screening may be performed by contacting a library ofthe invention with a target and isolating one or more library member(s)that bind to said target.

In another embodiment, individual members of said library are contactedwith a target in a screen and members of said library that bind to saidtarget are identified.

In another embodiment, members of said library are simultaneouslycontacted with a target and members of said library that bind to saidtarget are selected.

The target(s) may be a peptide, a protein, a polysaccharide, a lipid, aDNA or a RNA.

The target may be a receptor, a receptor ligand, an enzyme, a hormone ora cytokine.

The target ligand may be a prokaryotic protein, a eukaryotic protein, oran archeal protein. More specifically the target ligand may be amammalian protein or an insect protein or a bacterial protein or afungal protein or a viral protein.

The target ligand may be an enzyme, such as a protease.

It should be noted that the invention also embraces library member(s)isolated from a screen according to the invention. Suitably thescreening method(s) of the invention further comprise the step of:manufacturing a quantity of the complex of the invention isolated ascapable of binding to said targets.

The invention also relates to library members which are, or are capableof being, isolated by a screen according to the present invention,wherein said member is subsequently generated/manufactured without thefurther use of the nucleic acid which encoded said polypeptide when partof the complex of the invention. For example, the methods of theinvention suitable further comprise the additional step of manufacturinga quantity of a polypeptide isolated or identified by a method of theinvention by attaching the molecular scaffold to the polypeptide,wherein said polypeptide is recombinantly expressed or chemicallysynthesized. For example, when the polypeptide is recombinantlysynthesised in this embodiment, the nucleic acid originally encoding itas part of a complex of the invention may no longer be directly presentbut may have been present in an intermediate step eg. PCR amplificationor cloning of the original nucleic acid of the complex, leading toproduction of a template nucleic acid from which the polypeptide may besynthesised in this additional step.

Polypeptide ligands may have 1 2 or more more than two loops. Forexample, tricyclic polypeptides joined to a molecular scaffold can becreated by joining the N- and C-termini of a bicyclic polypeptide joinedto a molecular scaffold according to the present invention. In thismanner, the joined N and C termini create a third loop, making atricyclic polypeptide. This embodiment is suitably not carried out onphage, but is suitably carried out on a polypeptide-molecular scaffoldconjugate of the invention. Joining the N- and C-termini is a matter ofroutine peptide chemistry. In case any guidance is needed, theC-terminus may be activated and/or the N- and C-termini may be extendedfor example to add a cysteine to each end and then join them bydisulphide bonding. Alternatively the joining may be accomplished by useof a linker region incorporated into the N/C termini. Alternatively theN and C termini may be joined by a conventional peptide bond.Alternatively any other suitable means for joining the N and C terminimay be employed, for example N-C-cyclization could be done by standardtechniques, for example as disclosed in Linde et al. Peptide Science 90,671-682 (2008) “Structure-activity relationship and metabolic stabilitystudies of backbone cyclization and N-methylation of melanocortinpeptides”, or as in Hess et al. J. Med. Chem. 51, 1026-1034 (2008)“backbone cyclic peptidomimetic melanocortin-4 receptor agonist as anovel orally administered drug lead for treating obesity”. One advantageof such tricyclic molecules is the avoidance of proteolytic degradationof the free ends, in particular by exoprotease action. Another advantageof a tricyclic polypeptide of this nature is that the third loop may beutilised for generally applicable functions such as HSA binding, cellentry or transportation effects, tagging or any other such use. It willbe noted that this third loop will not typically be available forselection (because it is not produced on the phage but only on thepolypeptide-molecular scaffold conjugate) and so its use for other suchbiological functions still advantageously leaves both loops 1 and 2 forselection/creation of specificity. Thus the invention also relates tosuch tricyclic polypeptides and their manufacture and uses.

The present invention provides further methods for contacting thegenetically encoded compound libraries with a target ligand and foridentifying ligands binding to said target ligand. The geneticallyencoded compound libraries are assayed by either screening or selectionprocedures.

In a screening procedure, individual members of the library are assayed.Multiple copies of an individual member of the library are for exampleincubated with a target ligand. The target ligand is immobilized beforeor after contacting the members of the library and unbound members areremoved by washing. Bound ligands are for example detected in an enzymelinked immunosorbent assay (ELISA). The amino acid sequences of membersof the library that bind to the target ligand are determined bysequencing of the genetic code.

In a selection procedure, multiple members of the encoded compoundlibrary are contacted with one or more targets. The targets areimmobilized before or after contacting the members of the library andunbound members are removed by washing. The genetic code of boundligands is sequenced. Selected ligands are alternatively propagated toperform further selection rounds.

In one embodiment of the invention, the compound libraries are encodedby phage display and selections are performed by phage panning.

(iii) Phage Purification

Any suitable means for purification of the phage may be used. Standardtechniques may be applied in the present invention. For example, phagemay be purified by filtration or by precipitation such as PEGprecipitation; phage particles may be produced and purified bypolyethylene-glycol (PEG) precipitation as described previously.

In case further guidance is needed, reference is made to Jespers et al(Protein Engineering Design and Selection 2004 17(10):709-713. Selectionof optical biosensors from chemisynthetic antibody libraries.) Suitablyphage may be purified as taught therein. The text of this publication isspecifically incorporated herein by reference for the method of phagepurification; in particular reference is made to the materials andmethods section starting part way down the right-column at page 709 ofJespers et al.

Moreover, the phage may be purified as published by Marks et al J. Mol.Biol vol 222 pp 581-597, which is specifically incorporated herein byreference for the particular description of how the phageproduction/purification is carried out.

In case any further guidance is needed, phage may be reduced andpurified as follows. Approximately 5×10¹² phage particles are reactedwith 1 mM dithiothreitol (DTT) for 30 min at room temperature, then PEGprecipitated. After rinsing with water, the pellet is resuspended in 1ml of reaction buffer (10 mM phosphate buffer, 1 mM EDTA, pH 7.8). Thephage are then optionally reacted with 50 μl of 1.6 mMN-[(2-iodoacetoxy)ethyl]-N-methylamino-7-nitrobenz-2-oxa-1,3-diazole(NBDIA) (Molecular Probes) for 2 h at room temperature, or more suitablyreacted with the molecular scaffold as described herein. The reaction isterminated by PEG precipitation of phage particles.

A yet still further way in which the phage may be produced/purified isas taught in Schreier and Cortese (A fast and simple method forsequencing DNA cloned in the single-stranded bacteriophage M13. Journalof molecular biology 129(1):169-72, 1979). This publication deals withthe chain termination DNA sequencing procedure of Sanger et al. (1977),which requires single-stranded DNA as template. M13 phage DNA exists asa single strand and therefore every DNA sequence cloned in M13 can beeasily obtained in this form. Schreier and Cortese show that M13single-stranded DNA pure enough to be used as a template for sequencedetermination can be prepared by simple centrifugation of the phageparticle and extraction with phenol. The Schreier and Cortesepublication is specifically incorporated herein by reference for themethod of purification of the phage. For the avoidance of doubt, thephenol extraction is not performed for making complexes according to thepresent invention since that is for the purpose of nucleic acidpurification. Thus the phenol step of Schreier and Cortese is suitablyomitted. The Schreier and Cortese method is followed only to the pointof purified phage particles.

Thus there are myriad techniques well known in the art for purificationof phage. In the context of the present invention such purification isused for the removal of reducing agent used to reduce the reactivegroups in the polypeptide of interest for bonding to the molecularscaffold, particularly when such bonding is via cysteine residues.

Optionally, especially advantageous techniques for phage purificationmay be adopted as discussed in the reaction chemistry section below. Itshould be expressly noted that these techniques are not regarded asessential for the invention, but may represent especially helpfulmethods or even the best mode of making the phage particles of theinvention. However, provided attention is paid to avoiding reoxidationof the reduced functional/reactive groups on the phage at the stage ofremoval of the reducing agent before attachment of the molecularscaffold then in principle any technique may be used to accomplish this.The filtration techniques described are particularly effective but alsomore complicated than standard techniques so the operator will choosethe technique best suited to their particular working of the invention.Most suitably the filtration technique is employed.

(iv) Reaction Chemistry

Prior art technologies for modification of polypeptides have involvedharsh chemistry and independent polypeptide modification reactions. Bycontrast, the present invention makes use of chemical conditions for themodification of polypeptides which advantageously retain the functionand integrity of the genetically encoded element of the product.Specifically, when the genetically encoded element is a polypeptidedisplayed on the surface of a phage encoding it, the chemistryadvantageously does not compromise the biological integrity of thephage. It is disclosed herein that there is a narrow window ofconditions for which these chemical reactions can be enhanced orfacilitated. In particular, as will be explained in more detail below,the solvents and temperatures used are important to an efficientreaction. Furthermore, the concentration of the reagents used are alsoinstrumental in promoting the correct bonding, whilst ameliorating oreliminating cross linking or damaging of the polypeptide moieties whichare being modified.

In particular, it is disclosed that the reduction of the cysteines inthe target polypeptide is required for the most efficient reaction.Clearly, the reducing agent used to chemically reduce those cysteinesmust be removed in order to perform the desired attachment. One knowntechnique is to use dithiothreitol (DTT) or triscarboxyethylphosphine(TCEP) for reduction of the cysteines, and for the removal of thereducing agent to precipitate the particles such as the phage particlesin a precipitation reaction. Such precipitation reactions typicallyinvolve 20% polyethylene glycol (PEG) together with 2.5 molar NaCl whichleads to precipitation of the phage particles. However it is importantto avoid re-oxidation. As will be disclosed in more detail below, thesolutions are found in a range of strategies including the use ofdegassed buffer, the use of chelators in the reaction mixture, and theuse of filtration in order to extract the particles, or the use of lowconcentrations of TCEP in the presence of TBMB.

Reaction conditions e.g. for attachment of the molecular scaffold to thetarget polypeptide should be chosen carefully. Choice of conditions mayvary depending upon the application to which the invention is being put.Particular care is required when the target polypeptide is comprised bya phage particle. Guidance is provided throughout the specification andexamples section.

Reaction conditions as reaction temperature, molecular scaffoldconcentration, solvent and/or pH should be chosen to allow efficientreaction of the reactive groups of the target polypeptide with themolecular scaffold, but leave the nucleic acid encoding the polypeptidein a condition that allows to decode (e.g. to sequence) and/or propagatethe isolated molecules (e.g. by PCR or by phage propagation or any othersuitable technique). Moreover, the reaction conditions should leave thephage coat protein in a condition that allows it to propagate the phage.

Thiol groups of a phage encoded polypeptide may be reduced with reducingagent prior to molecular scaffold attachment. In such embodiments, inparticular in phage display embodiments, or in particular when thereducing agent is TCEP, the excess of reducing agent is suitably removedby filtration e.g. filtration of the phage. This is especiallyadvantageous since the present inventors disclose for the first timethat conventional techniques for removal of reducing agents such asPEG/NaCl precipitation can sometimes lead to sub-optimal reaction withmolecular scaffold, likely due to reoxidation of the reduced functionalside groups of the target polypeptide. Thus it is an advantage ofembodiments in which the target polypeptide is prepared by reductionfollowed by purification (removal of reducing agent) via filtration thatsuperior preservation of the reduced (and hence reactive) reactivegroups of the polypeptide is achieved.

In the present invention, reaction conditions are applied that on theone hand allow to efficiently link the encoded polypeptide to amolecular scaffold and on the other hand leave the appended nucleic acid(and phage coat proteins) in a condition that allows its propagation ordecoding. Said reaction conditions are for example the reactiontemperature, molecular scaffold concentration, solvent composition orpH.

In one embodiment of the present invention, thiol groups of cysteineresidues are used as reactive groups to link polypeptides to a molecularcore. For some chemical reactions, the thiol groups of the polypeptidesneed to be reduced. Thiol groups in phage displayed polypeptides areefficiently reduced by addition of a reducing agent as for exampletris(carboxyethyl)phosphine (TCEP). Since an excess of reducing agentcan interfere with the attachment reaction it is largely removed byfiltration of the phage, or by PEG precipitation, although lowconcentrations (10 micromolar or less) may be desirable to maintainreducing conditions during the attachment reaction.

Re-oxidation of the thiol groups after removal of TCEP is suitablyprevented by degassing of the reaction buffer.

Re-oxidation of the thiol groups is also suitably prevented by complexformation of metal ions by chelation, for example chelation withethylenediaminetetraacetic acid (EDTA).

Most suitably re-oxidation of the thiol groups is prevented or inhibitedby both chelation and use of degassed buffers.

In one embodiment of the present invention, attachment of thepolypeptide to the molecular scaffold is accomplished by reacting thereactive groups of the polypeptide such as thiol groups of a phageencoded polypeptide with the molecular scaffold for one hour.

Suitably they are reacted at 30° C.

Suitably they are reacted with molecular scaffold (such astris(bromomethyl)benzene) at a concentration of 10 μM.

Suitably reaction is in aqueous buffer.

Suitably reaction is at pH 8.

Suitably reaction buffer contains acetonitrile. Suitably reaction buffercontains 20% acetonitrile.

Most suitably the reaction features two or more of the above conditions.Suitably the reaction features three or more of the above conditions.Suitably the reaction features four or more of the above conditions.Suitably the reaction features five or more of the above conditions.Suitably the reaction features six or more of the above conditions.Suitably the reaction features each of the above conditions.

These reaction conditions are optimized to quantitatively react thiolgroups of a polypeptide with the reactive groups oftris(bromomethyl)benzene. Under the same reaction conditions, about 20%of the phage particles remain infective to bring the genetic code intobacterial cells for propagation and decoding.

In one embodiment the molecular scaffold, such as TBMB, may be attachedto the target polypeptide, such as a phage encoded polypeptide, byreaction (incubation) of thiol groups of the polypeptide for one hour at30° C. with TBMB (i.e. tris(bromomethyl)benzene) at a concentration of10 μM in aqueous buffer pH 8 containing 20% acetonitrile.

The inventors also disclose the effect of concentration of the molecularscaffold in the reaction on phage infectivity. In particular theinvention suitably minimises the concentration of molecular scaffoldused in the reaction. In other words, the lower the concentration ofmolecular scaffold used at the time of reaction with the polypeptide ofthe phage, the better, provided always that sufficient molecularscaffold becomes joined to the phage polypeptide. The advantage ofminimising the molecular scaffold present in this way is superiorpreservation of phage infectivity following coupling of the molecularscaffold. For example, when the molecular scaffold is TBMB,concentrations of molecular scaffold greater than 100 μM can compromiseinfectivity. Thus suitably when the molecular scaffold is TBMB thensuitably the concentration of TBMB present at the time of bonding to thepolypeptide is less than 100 μM.

(C) Use of Peptide Ligands According to the Invention

Peptide ligands selected according to the method of the presentinvention may be employed in in vivo therapeutic and prophylacticapplications, in vitro and in vivo diagnostic applications, in vitroassay and reagent applications, and the like.

In general, the use of a peptide ligand can replace that of an antibody.Ligands selected according to the invention are of use diagnostically inWestern analysis and in situ protein detection by standardimmunohistochemical procedures; for use in these applications, theligands of a selected repertoire may be labelled in accordance withtechniques known in the art. In addition, such polypeptide ligands maybe used preparatively in affinity chromatography procedures, whencomplexed to a chromatographic support, such as a resin. All suchtechniques are well known to one of skill in the art. Peptide ligandsaccording to the present invention possess binding capabilities similarto those of antibodies, and may replace antibodies in such assays.

Diagnostic uses include any uses which to which antibodies are normallyput, including test-strip assays, laboratory assays and immunodiagnosticassays.

Therapeutic and prophylactic uses of peptide ligands prepared accordingto the invention involve the administration of ligands selectedaccording to the invention to a recipient mammal, such as a human.Substantially pure peptide ligands of at least 90 to 95% homogeneity arepreferred for administration to a mammal, and 98 to 99% or morehomogeneity is most preferred for pharmaceutical uses, especially whenthe mammal is a human. Once purified, partially or to homogeneity asdesired, the selected polypeptides may be used diagnostically ortherapeutically (including extracorporeally) or in developing andperforming assay procedures, immunofluorescent stainings and the like(Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes Iand II, Academic Press, NY).

The peptide ligands of the present invention will typically find use inpreventing, suppressing or treating inflammatory states, allergichypersensitivity, cancer, bacterial or viral infection, and autoimmunedisorders (which include, but are not limited to, Type I diabetes,multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus,Crohn's disease and myasthenia gravis).

In the instant application, the term “prevention” involvesadministration of the protective composition prior to the induction ofthe disease. “Suppression” refers to administration of the compositionafter an inductive event, but prior to the clinical appearance of thedisease. “Treatment” involves administration of the protectivecomposition after disease symptoms become manifest.

Animal model systems which can be used to screen the effectiveness ofthe peptide ligands in protecting against or treating the disease areavailable.

Methods for the testing of systemic lupus erythematosus (SLE) insusceptible mice are known in the art (Knight et al. (1978) J Exp. Med.,147: 1653; Reinersten et al. (1978) New Eng. J: Med., 299: 515).Myasthenia Gravis (MG) is tested in SJL/J female mice by inducing thedisease with soluble AchR protein from another species (Lindstrom et al.(1988) Adv. Inzn7unol., 42: 233). Arthritis is induced in a susceptiblestrain of mice by injection of Type II collagen (Stuart et al. (1984)Ann. Rev. Immunol., 42: 233). A model by which adjuvant arthritis isinduced in susceptible rats by injection of mycobacterial heat shockprotein has been described (Van Eden et al. (1988) Nature, 331: 171).Thyroiditis is induced in mice by administration of thyroglobulin asdescribed (Maron et al. (1980) J. Exp. Med., 152: 1115). Insulindependent diabetes mellitus (IDDM) occurs naturally or can be induced incertain strains of mice such as those described by Kanasawa et al.(1984) Diabetologia, 27: 113. EAE in mouse and rat serves as a model forMS in human. In this model, the demyelinating disease is induced byadministration of myelin basic protein (see Paterson (1986) Textbook ofImmunopathology, Mischer et al., eds., Grune and Stratton, New York, pp.179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al.(1987) J; Immunol., 138: 179).

Generally, the present peptide ligands will be utilised in purified formtogether with pharmacologically appropriate carriers. Typically, thesecarriers include aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, any including saline and/or buffered media. Parenteralvehicles include sodium chloride solution, Ringer's dextrose, dextroseand sodium chloride and lactated Ringer's. Suitablephysiologically-acceptable adjuvants, if necessary to keep a polypeptidecomplex in suspension, may be chosen from thickeners such ascarboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous vehicles include fluid and nutrient replenishers andelectrolyte replenishers, such as those based on Ringer's dextrose.Preservatives and other additives, such as antimicrobials, antioxidants,chelating agents and inert gases, may also be present (Mack (1982)Remington's Pharmaceutical Sciences, 16th Edition).

The polypeptide ligands of the present invention may be used asseparately administered compositions or in conjunction with otheragents. These can include antibodies, antibody fragments and variousimmunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycinor cisplatinum, and immunotoxins. Pharmaceutical compositions caninclude “cocktails” of various cytotoxic or other agents in conjunctionwith the selected antibodies, receptors or binding proteins thereof ofthe present invention, or even combinations of selected polypeptidesaccording to the present invention having different specificities, suchas polypeptides selected using different target ligands, whether or notthey are pooled prior to administration.

The route of administration of pharmaceutical compositions according tothe invention may be any of those commonly known to those of ordinaryskill in the art. For therapy, including without limitationimmunotherapy, the selected antibodies, receptors or binding proteinsthereof of the invention can be administered to any patient inaccordance with standard techniques. The administration can be by anyappropriate mode, including parenterally, intravenously,intramuscularly, intraperitoneally, transdermally, via the pulmonaryroute, or also, appropriately, by direct infusion with a catheter. Thedosage and frequency of administration will depend on the age, sex andcondition of the patient, concurrent administration of other drugs,counter-indications and other parameters to be taken into account by theclinician.

The polypeptide ligands of this invention can be lyophilised for storageand reconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective and art-known lyophilisation andreconstitution techniques can be employed. It will be appreciated bythose skilled in the art that lyophilisation and reconstitution can leadto varying degrees of activity loss and that use levels may have to beadjusted upward to compensate.

The compositions containing the present polypeptide ligands or acocktail thereof can be administered for prophylactic and/or therapeutictreatments. In certain therapeutic applications, an adequate amount toaccomplish at least partial inhibition, suppression, modulation,killing, or some other measurable parameter, of a population of selectedcells is defined as a “therapeutically-effective dose”. Amounts neededto achieve this dosage will depend upon the severity of the disease andthe general state of the patient's own immune system, but generallyrange from 0.005 to 5.0 mg of selected peptide ligand per kilogram ofbody weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonlyused. For prophylactic applications, compositions containing the presentpolypeptide ligands or cocktails thereof may also be administered insimilar or slightly lower dosages.

A composition containing a polypeptide ligand according to the presentinvention may be utilised in prophylactic and therapeutic settings toaid in the alteration, inactivation, killing or removal of a selecttarget cell population in a mammal. In addition, the selectedrepertoires of polypeptides described herein may be usedextracorporeally or in vitro selectively to kill, deplete or otherwiseeffectively remove a target cell population from a heterogeneouscollection of cells. Blood from a mammal may be combinedextracorporeally with the selected peptide ligands whereby the undesiredcells are killed or otherwise removed from the blood for return to themammal in accordance with standard techniques.

EXAMPLES

In the following examples, we describe alterations of individual membersof a first repertoire of peptide ligands; similar methods can be usedmake alterations of entire repertoires. In some cases it may be possibleto use similar chemistry and library format for making both repertoires.Thus a first repertoire of peptide ligands conjugated through cysteineresidues with trisbromomethylbenzene can be altered according to theinvention to give a second repertoire of the same peptides conjugatedwith trisbromomethylmesitylene under similar conditions. Furthermore asthe chemistry and reaction conditions are suitable for phage display,both first and second repertoire can be displayed and selected on phage.

However alterations made by conjugation with other scaffolds, forexample those reacting through iodoacetyl or acryloyl moieties of thescaffold, although similar for making synthetic peptides may not besuitable for phage display; if not it will be necessary to use adifferent library format. One format that is tolerant to greater rangeof chemistries and more extreme reaction conditions is the syntheticpeptide array. For reference see for example Min & Mrksich. CurrentOpinion in Chemical Biology, Volume 8, Issue 5, October 2004, Pages554-558. This format is particularly convenient if the selection factorrequired (and therefore library size) is not large, as the number ofpeptides that can arrayed conveniently are in the hundreds or thousands,rather than the millions or billions that can be selected by phagedisplay. It is therefore suitable for refinement of the properties oflead peptides, for example for improving resistance to protease, orbinding affinity to target.

The synthetic peptide array is also a highly suitable format forreacting with multiple scaffold species; in this way the same set ofpeptides can be used with different scaffold species to increase thestructural diversity of the second repertoire. The use of syntheticpeptides is also highly suitable for alterations of the reactive groupof the peptide to those not normally incorporated during biologicalpeptide synthesis, for example the alteration of cysteine tohomocysteine.

If phage display can be used for creation of both first and secondrepertoires, it is not necessary (or convenient) to determine thesequence of each member of the first repertoire in order to make thealterations required to generate the second repertoire. However, ifhowever phage display is used for the first repertoire, and a syntheticpeptide array for the second repertoire, it will be necessary todetermine the polypeptide sequences of the first repertoire in order tospecify those of the second repertoire. In this case the phage nucleicacid encoding the region of each of the displayed peptide conjugates canbe readily sequenced.

The invention is further described with reference to the followingexamples.

EXAMPLES Example 1 Protease Resistant Bicyclic Peptide Against MDM2

MDM2 is an enzyme (an E3 ubiquitin ligase) that recognises thetrans-activation domain of p53, the tumour suppressor, leading toubiquitinylation and degradation of p53 by the proteasome. A nutlininhibitor of the p53-MDM2 interaction can lead to in vivo activation ofthe p53 pathway, and it has been suggested that such agents may havepotential as anti-cancer agents. Here we describe the selection of twobicyclic peptides (PEP10 and PEP48) against MDM2, a target “antigen”.The affinity of each synthetic peptide was in the range 250-750 nM.

Protocols generally followed those described earlier in Heinis et al.,2009, Nature Chemical Biology 5, 502-507, unless otherwise indicated. Inthe work of Heinis et al., both targets, kallikrein and cathepsin G,were proteases, and the kallikrein inhibitor is fairly resistant toproteolysis by kallikrein, although it includes a kallikrein cleavagesite. MDM2 is not a protease, and therefore it was not clear whether theselected peptides would also be resistant to protease. For this, andother reasons (for detail see below), we included one or more protease(chymotrypsin) steps after reaction of the phage peptide repertoireswith the TBMB (including under reducing conditions) and before selectionof the repertoire against MDM2. The two selected phage peptides PEP10and PEP 48 appear resistant to proteolysis, as shown by phage ELISA.

Phage Production and Purification

The phage peptide library with diversity of at least 4×10⁹ clones wasprepared and TBMB conjugated as described earlier with a fewmodifications.

-   -   1. The cx6 library of phage as described earlier (which had been        prepared from TG1 cells) was used to infect the non-suppressor        strain HB2151 (Carter, Bedouelle & Winter. 1985. Nucleic Acids        Res. 13:4431-43), and the infected cells plated. The bacteria        were scraped from the plates in about 8 ml 2×TY medium, 30 ug/ml        chloramphenicol, 10% glycerol (v/v).    -   2. About 0.8 ml of the stock was added to 800 ml 2×TY medium        with 30 ug/ml chloramphenicol to obtain an OD of about 0.1 at        600 nm. The culture was incubated at 30° C., and shaken in a 2        litre flask at 200 rpm for 16 hrs.    -   3. The cell culture was centrifuged at 4,000 rpm (Heraeus        Megafuge 2R) for 30 min at 4° C. The supernatant was transferred        to 200 ml cold 20% PEG, 2.5 M NaCL. The mixture was left on ice        for 1 hr.    -   4. The precipitated supernatant/phage mixture was spun down for        30 min at 4° C. and the supernatant was discarded.    -   5. The phage was resuspended in 35 ml PBS, 5 mM EDTA followed by        spinning for 15 min at 4000 rpm (Heraeus Megafuge 2R) to remove        cellular debris. The supernatant was transferred into a new 50        ml Falcon tube.

Modification of Phage with TBMB

-   -   1. 5 ml of 8 mM TCEP (in H₂O) was added to the phage to obtain a        final concentration 1 mM TCEP. The tube was inverted several        time to mix and incubated for 1 hr at 42° C. water bath.    -   2. The TCEP was removed by a second PEG precipitation. 10 ml of        20% PEG, 2.5 M NaCL (degassed solution) was added, mixed, and        incubated on ice for 45 min and spun for 30 min at 4000 rpm, 4°        C.    -   3. The supernatant was carefully removed and pellet resuspended        in 12 ml PBS, 5 mM EDTA, 10 uM TCEP (degassed buffer)    -   4. 3 ml of 50 uM TBMB in acetonitrile was added to the 12 ml of        reduced phage to obtain a final TBMB concentration of 10 uM. The        tube was inverted several times and left at 30° C. for 1 hr in a        water bath. The phage were cooled on ice and precipitated with        1/5 volume of 20% PEG, 2.5 M NaCL for 30 min. The phage were        collected by spinning at 4000 rpm (Hereaus Megafuge 2R) for 20        min. Supernatant was removed and the phage resuspended in 4 ml        of PBS. Phage was transferred into the 2 ml Eppendorf tubes and        spun at 13000 rpm (Eppendorf benchtop centrifuge) for 10 min.        Supernatant was transferred into a new Eppendorf tube and phage        infectivity was measured.

Phage Selection: General Protocol

First Round of Selection

-   -   1. Purified and chemically conjugated phage as above was        selected against biotinylated MDM2 (bio-MDM2) peptide (res        2-125) immobilized on the surface of the streptavidin-coated        Dynabeads (Dynal Biotech). 80 μl of beads were first washed and        blocked with 2% (w/v) Marvell milk powder in PBS (PBSM) for 40        min followed by incubation with 100 nM bio-MDM2 for 20 min in a        total volume of 1 ml.    -   2. Chemically modified phage (10¹⁰-10¹¹TU) was incubated with        PBSM for 40 min.    -   3. Blocked Ag-coated beads from step 1 were washed from the        excess of the Ag with 0.1% Tween in PBS (PBST) and incubated        with the blocked phage for 30 min in a total volume of 1 ml.    -   4. Unbound phage were washed with 10× with PBST followed by 2×        with PBS. After each third washing step the phage coated beads        were transferred into a new Eppendorf tube.    -   5. Phage were eluted by incubating with 500 μl of 50 mM glycine        pH 2.2 for 10 min on a rotating wheel. Eluted phage were        neutralized with 250 μl of 1M Tris, pH7.5.    -   6. 375 μl of phage was incubated with 10 ml of HB2151 cells for        90 min at 37° C. without shaking.    -   7. The infected cells were then shaken for 30 min at 37° C. and        then plated on a chloramphenicol plate (20×20 cm).    -   8. The colonies were scraped off the plate in 2×TY,        chloramphenicol, 10% glycerol as described above, and stored as        a glycerol stock at −80° C. A fraction of the cells was used to        prepare phage for the second round of selection.

Second Round of Selection

The second round of selection was similar to the first one except for afew modifications.

-   -   1. Neutravidin-coated magnetic beads were used instead of        streptavidin ones.    -   2. The amount of antigen used in the selection was 20 nM.    -   3. Chemically modified phage (10¹⁰-5×10¹⁰ TU) was first treated        with 50 ug/ml of chymotrypsin for 2 min followed by blocking        with PBSM for 40 min.    -   4. Unbound phage was washed with 15× with PBST followed by 2×        with PBS, otherwise as above.

Phage Selection: Variant Protocol

Clone 48 was selected using the general protocol as above, whereas clone10 was developed as a result of a modified protocol being introduced.The modifications are the following:

-   -   1. In the first round chemically modified phage were pre-treated        with 50 ug/ml of chymotrypsin for 2 min followed by blocking        with PBSM for 40 min.    -   2. In the second round chemically modified phage were first        reduced with 5 mM DTT for 20 min followed by incubation with 50        ug/ml of chymotrypsin for 2 min and blocking with PBSM for 40        min.

Peptide Synthesis

The encoded peptides from phage clone 48 and phage clone 10 weresynthesized with free N- and C-termini. PEP10:H-Ser-Cys-Glu-Leu-Trp-Asn-Pro-Lys-Cys-Arg-Leu-Ser-Pro-Phe-Glu-Cys-Lys-Gly-OH(SEQ ID No. 5); PEP48:H-Ser-Cys-Val-Arg-Phe-Gly-Trp-Thr-Cys-Asp-Asn-Ser-Trp-His-Gly-Cys-Lys-Gly-OH(SEQ ID No. 6).

The syntheses were performed employing Fmoc chemistry on a CEM Libertymicrowave peptide synthesizer at a 0.1 mmol scale. Fmoc-Gly-PEG PS resinusing a 5-fold excess of Fmoc-amino-acids activated with PyBOP in DMFand DIPEA in NMP (1 equivalent and 2 equivalents respectively.Side-chain protecting groups were as follows: Arg(Pbf); Asn(Trt);Asp(OtBu); Cys(Trt); Glu(OtBu); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc).Fmoc-deprotection was carried out using 20% v/v Piperidine/DMFcontaining 0.1 M HOBt. The H-peptidyl-resins were washed with DMF, thenpropan-2-ol and dried in vacuo. Cleavage of side-chain protecting groupsand from the support was effected using 94:2.5:2.5:1 v/v/v/vTFA/EDT/H₂O/iPr₃SiH for 2 hours. The peptide/TFA mixture was filtered toremove the support and the peptide/TFA mixture was diluted with waterand washed with Et₂O (5-times) and the aqueous layer lyophilized.

Reverse-phase HPLC was performed on a Phenomenex Jupiter 5p C18 300 Å250×4.6 mm column. Buffer A: 0.1% TFA/H20; Buffer B: CH3CN containing10% Buffer A. The column was eluted isocratically with 10% Buffer B for2 minutes, then with a linear gradient of 10-90% over 25 minutes.Detection was at 215/230 nm; flow rate of 1.5 ml/min.

The peptides were lyophilized and checked by mass spectrometry. PEP10MALDI-TOF mass (M+H): 2099.9 Da (Theory: 2098.4 Da.) PEP48 MALDI-TOFMass (M+H): 2043.8 Da (Theory: 2042.8 Da.). The peptides were thenconjugated with TBMB.

Synthesis of TBMB-Peptide Conjugates

Initial reactions were performed to mimic the conditions used duringphage selection. Typically, 5 mg of the purified peptide was dissolvedin 1 ml water and 0.8 ml 50 mM NH₄HCO₃ added, followed by enough TCEP tobring the final concentration to 10 m TCEP. TBMB (3 equivalents based onweight of peptide) dissolved in MeCN was added to the reaction. Thereaction was left for 1.5 hrs then monitored by HPLC. On completion thereaction was purified by HPLC. Typically 0.5 to 1.5 mg of final productwas obtained. However this method gives rise to several by-products, themajor one having an additional mass of +250 Dalton. This corresponds toaddition of TCEP to the desired product, and the yield of this productincreases with reaction time. In addition, other higher mass productscorresponding to addition of a second TBMB were observed by MALDI-TOFmass spec, but were not isolated.

Based on the formation of TCEP adducts a preferred method was developed.Following cleavage of the peptide from the resin, it was either purifieddirectly by HPLC or pre-treated with TCEP/DTT for 15 mins prior to HPLCpurification. The product from the HPLC purification, in the HPLCelution buffer (typically 6 ml) is neutralised with 50 mM NH₄HCO₃ (4 ml)and TBMB added in MeCN as above. The addition of 10% THF results in aclear solution and therefore accelerates the reaction. Reactions aremonitored by mass spec, but typically are complete in 1-2 hrs. There areminimal by-products from this reaction (though the presence of product+16 is observed by mass spec). The reaction requires concentration toremove organic solvents prior to HPLC purification otherwise the producttends to elute with the solvent front. Yields of product from thismethod are typically 0.5 to 1.5 mg from 3 mg peptide, but this has notbeen optimised.

Binding Assays

Phaqe ELISA Assay

0.6 μg/mL of biotinylated MDM2 peptide (res 2-125) was immobilized on astreptavidin-coated plate (Roche). Plate was blocked with PBSM (but 4%in milk powder) and linear or TBMB-conjugated phage (10⁷ TU/well in PBSMin the presence or absence of 5 mM DTT) was incubated on the plate for50 min at room temperature. Similarly, phage was first reduced in 5 mMDTT for 20 min, treated with chymotrypsin (50 ug/ml in PBS) for 2 min,mixed with PBSM (final concentration) and incubated on the plate for 50min at room temperature. Phage was detected using an anti-M13-HRPmonoclonal antibody (1:5000, Amersham).

The results showed qualitatively that both phage clones 10 and clone 48bind to MDM2 as the cyclic conjugate but not as the unconjugated peptide(whether or not pre-treated with DTT). Furthermore the binding of theconjugated peptide resists proteolysis. Note that 5 mM DTT can reducethe disulphide bonds of chymotrypsin leading to its inactivation as aprotease. To ensure that the chymotrypsin was active under theconditions of the assay, we incubated control phage bearing a linearpeptide that binds MDM2 after pre-treatment as above with 5 mM DTT.Under the conditions of our experiment, the binding activity of thecontrol phage was lost on proteolysis. In other experiments we have usedup to 0.2 mM-5 mM TCEP in the presence of chymotrypsin (0.1 mg/ml-1mg/ml) for 2 minutes at room temperature in PBS. These conditions alsoallowed us to distinguish between the linear and cyclic peptides onphage.

Fluorescence Anisotropy Measurements

Titration experiments were run on a Horiba Jobin Yvon fluorimeterequipped with the Hamilton Microlab titrator controlled by laboratorysoftware. The λ_(ex) and λ_(em) used were 295 nm and 350 nm,respectively. The slit widths for excitation and emission were 5 nm and15 nm and the integration time 1 Os was used for each measurement. Theintrinsic fluorescence of tryptophan in peptides 10, 48 was used tomeasure their binding affinity for MDM2 (res 2-125). The experimentswere performed at 23° C. in PBS, 5 mM DTT. Usually 250 μl of MDM2 (150micromolar) was titrated into 1.2 ml of peptide (1 micromolar).Titration data were analyzed with a standard 1:1 binding model by usingthe quadratic solution to the equilibrium Kd=[A][B]/[AB]. Kd is thedissociation rate, and [A] and [B] refer to the concentration of atitrant (MDM2) and fluorescent peptides 10 and 48, respectively. Thefitting equation contained an extra term to account for linear drift.

The results (below) indicate that the affinity of each peptide issub-micromolar, and in the range 250-750 nM. The measurments for PEP48were repeated.

PEP10+MDM2, measured λex=295 nm, Kd=267 nM;

PEP48+MDM2, measured λex=280 nm, Kd=760 nM;

PEP48+MDM2, measured λex=295 nm, Kd=567 nM

Competition Assays

The binding of PEP48 phage to MDM2 was competed by a peptide pMI(TSFAEYWNLLSP (SEQW ID No. 7)) that binds to MDM2 at the p53 site with aKd=3.3 nM (Pazgier et. al., 2009 PNAS, 106, 4665-4670). 0.6 μg/ml ofbiotinylated MDM2 peptide (res 2-125) was immobilized on astreptavidin-coated plate (Roche). Plate was blocked with PBSM.TBMB-conjugated phage (10⁷ TU/well in 1% PBSM) was premixed with a rangeof concentrations of pDI (from 6.94 nM to 1 uM) and incubated on theplate for 75 min at room temperature. Phage was detected using ananti-M13-HRP monoclonal antibody (1:5000, Amersham). The binding ofPEP48 phage to MDM2 was inhibited by addition of the pMI peptide, withan estimated 1050=125 nM.

Example 2 Oxidation of PK15-TBMB Conjugate to its Sulfoxide and Sulfone

PK15-TBMB was synthesised as described in Heinis et al., 2009, NatureChemical Biology 5, 502-507. Approximately 1 mg of PK15-TBMB wasdissolved in 1 ml of 1×PBS and hydrogen peroxide added to aconcentration of 0.3%. Reaction left at room temperature overnight.MALDI mass spec showed a range of peaks corresponding to the addition of1,2 and 3 oxygen atoms, but incomplete reaction. Reaction adjusted to 1%H₂O₂ and left for 8 hrs where it could be seen that the +3 (oxygen)product was major but the reaction still incomplete. The reaction washeated in a microwave synthesiser, initially cooled on ice, up to atemperature of 37° C., with a power of up to 50 W. After 15 mins massspec showed essentially a single peak at 1992 corresponding to additionof 3 oxygen atoms, and therefore corresponding to the sulphoxides. Thereis virtually no sign of addition of further oxygen atoms under theseconditions. HPLC showed essentially a single peak.

The oxidised derivative was compared for its ability to inhibitkallikrein.

Enzymes were purchased from Sigma Aldrich and substrates from Bachem AG.The assay buffer is composed of 10 mM Tris pH 7.4, 150 mM NaCl, 10 mMMgCl₂, 1 mM CaCl₂, 0.1% BSA, 0.01% Triton X100 and 5% DMSO. Enzymes areincubated with inhibitors for 30 minutes at RT prior to addition ofsubstrate. All experiments were recorded at 30° C. for 90 minutes.

Assays were performed on a BMG Pherastar plate reader at wavelengths ofexc/em 350/450 nm. Kallikrein was bought as a solution of 1080 μg/mL anddiluted to a working concentration of 0.3 nM in assay buffer. SubstrateZ-Phe-Arg-AMC was solubilised at the stock concentration of 10 mM inDMSO and diluted to a working concentration of 300 μM with assay buffer.Inhibitors were solubilised in assay buffer to a stock concentration of60 μM. 50 μL of each reagent is introduced in wells for a final volumeof 150 μL per well. Final concentration of kallikrein in assay is 0.1 nMand substrate is 100 μM.

Final concentrations of inhibitors were: 0.5 nM, 1 nM, 2 nM, 5 nM, 8 nM,10 nM, 20 nM, 50 nM, 80 nM, 100 nM, 200 nM, 500 nM, 800 nM, 1 μM, 2 μM,5 μM, 8 μM, 10 μM and 20 μM. The initial rate of the reaction isobtained by plotting fluorescence=f (time) data and by fitting a lineartrendline for each concentration of inhibitor. The inhibition curves areobtained by plotting initial rate=f ([I]) and IC₅₀ values can beevaluated.

Under conditions where PK15-TBMB inhibited kallikrein with an 1050 of 13nM, preliminary results indicated that the oxidized PK15-TBMB inhibitedkallikrein with an 1050 of about 2.4 μM. Thus the change in the natureof the attachment of core to the peptide had a dramatic effect on theaffinity of the ligand. Note that the formation of sulphoxides at threesites is expected to lead to stereoisomers of the constrained peptide asthe oxidation with a single atom of oxygen creates a chiral centre atthe sulphur atom. The stereoisomers are likely to differ in theirinteractions with the target, and the IC50 value is therefore an averageof the IC50 values for each of the stereoisomers.

Subsequently conditions were established for oxidation of the conjugatesto the sulphone. PK15-TBMB (4 mg) was dissolved in 1 ml water andmagnesium monoperoxy phthalate (10 equivalents, 10 mg in 0.5 ml water)added. The reaction was monitored by MALDI-TOF MS until completion ofreaction in 45 min and then purified by HPLC. Yield 1.8 mg, molecularweight found 2038 (calculated 2038). The product was purified by HPLC.Note that in this case, the sulphur is no longer a chiral centre, andtherefore a single molecular species is expected. This oxidizedPK15-TBMB inhibited kallikrein with an 1050 of 1.3 micromolar. Theoxidation of the sulphur is expected to change the bond angles subtendedaround the sulphur, and also the packing of atoms around the core. Botheffects could be responsible for the alteration of binding affinity.

Example 3 Use of Trimethylmesitylene and Triethylmesitylene Cores

PK15 was conjugated with tris(bromomethyl)mesitylene under similarconditions to those described earlier with tris(bromomethyl)benzene(Heinis et al., 2009), and purified by HPLC. The conjugate was comparedfor its ability to inhibit kallikrein according to the procedure setforth in Example 1. Under conditions where PK15-TBMB inhibitedkallikrein with an 1050 of 13 nM, preliminary results indicated that thehexamethyl benzene conjugate inhibited kallikrein with an 1050 of 150nM. PK15 was also conjugated with tris(bromoethyl)mesitylene as above;this led to a further loss of inhibition, with an 1050 of about 400 nM.Thus the change in the nature of the core had a dramatic effect on theaffinity of the ligand. In this case, the extra bulk of the three methylor ethyl groups will likely have altered the packing around the core,and therefore the binding affinity.

Example 4 Use of Other Scaffold Cores Based on Iodoacetyl (IAc) andAcryloyl (Acr) Functionalities

All scaffold precursor structures are shown in Table 1. They were thenmodified with thiol-reactive functionalities to yield the finalscaffold, also as shown in Table 1.

The scaffold precursors trishydroxymethylethane (for THME(IAc3)),pentaerythritol (for PE(IAc4)), triethanolamine (for TEA(IAc3)), styreneR-epoxide (for TPEA(IAc3)), tris(4-formylphenyl)amine (for TPBA(IAc3)),salicylamide (TOHPT(IAc3)), cyanuric chloride and Boc-piperazine (forTPT(Acr3)) were acquired from Sigma Aldrich.

The synthesis of the precursor for TPEA(IAc3) was performed through thecondensation of styrene R-epoxide with ammonia in methanol (2 M, Sigma),using microwave irradiation for enhanced reaction rates, as described inFavretto et al, and Nugent et al (Tet. Let., 2002, 43, 2581-2584; and J.Am. Chem. Soc., 1994, 116, 6142-6148, respectively). Purification of thecrude (R,R,R)-tris(2-phenyl)-ethanolamine product was performed usingflash column chromatography and TLC as described.

The precursor for TPBA(IAc3) (tris-(4-hydroxymethylphenyl)amine) wasprepared from the commercial aldehyde (tris(4-formylphenyl)amine, 0.1mmole) by reduction with NaBH₄ in 20 mL THF (1 hr at RT), quenched with1 mL HAc (100%), and phase extracted with DCM/H₂O. The resultanttris-(4-hydroxymethylphenyl)amine was kept in DCM solution for furthermodification (see below).

The precursor tris(ortho-hydroxyphenyl)triazine was obtained by thecondensation of salicylamide at 270° C. for 3 hours, as described inJohns et al (J. Org. Chem., 1962, 27 (2), pp 592-594) and Cousin et al(Bull. Soc. Chim. France 1914, (4) 15, 416). The crude yellow product (3g) was solubilised in concentrated NaOH (aq), and precipitated withdilute sulphuric acid. The yellow precipitate was collected bycentrifugation, and repeatedly washed with H₂O and EtOH, then DCM, anddried. The product was then taken up in hot DMF, which upon cooling to−20° C. precipitated tris(ortho-hydroxyphenyl)triazine as small darkyellow crystal clusters.

The Boc-protected precursor for TPT(Acr3) (tris-(Boc-piperazine)-1,3,5triazine) was prepared from cyanuric chloride and N-boc piperazine asdetailed by Chouai et al (J. Org. Chem., 2008, 73 (6), pp 2357-2366).Boc removal was achieved with 6N HCl in MeOH for 12 hrs. The crudetris-piperazino-triazine was purified by standard acid/base extraction,followed by phase extraction using DCM/H₂O as solvents.

Iodoacetyl and Acryloyl groups are well-established and selectivethiol-reactive groups (Hermanson, Bioconjugate Techniques 2^(nd)edition, Academic Press, 2008). The advantage of using these groups isthat they do not require an aromatic system nearby (as is the case withTBMB), thus broadening the scope and chemical space available forthiol-reactive scaffolds.

All scaffold precursors in Table 1 contain hydroxyl groups (or secondaryamino groups in the case of the precursor for TPT(Acr3)). Introductionof the iodoacetyl thiol-reactive functionality onto the hydroxyl isachieved by the following two-step approach:

(Reaction of chloro-acetylchloride with an alcohol)

The purified product is then subjected to halogen exchange (Finkelsteinreaction) to obtain the final iodoacetylated product:

The reaction with chloro-acetylchloride according to Eq1 was usuallydone with ˜0.1 mmole scaffold precursor dissolved in 15 mL anhydrousTHF. After addition of 4 equivalents of pyridine and 3.5 equivalents ofchloro-acetylchloride (Sigma Aldrich), the reaction was allowed toproceed for 1-2 hours, at RT. The resultant THF-insoluble pyridine-HClwas then removed by centrifugation, and remaining chloro-acetylchloride(in theory, 0.5 equivalents) was quenched by the addition of 0.5 mL ofsaturated NaHCO₃ in H₂O. After rotaevaporation of the solvent, the oilyproduct was taken up in ˜10 mL acetone, and 5 equivalents of NaI(pre-dissolved at 1 M in acetone) were added (Eq2). This was reacted forup to four hours at RT. As the reaction proceeds, NaCl is precipitated,and a cessation of NaCl precipitation indicated complete replacement ofall chlorines with iodines. Solvent was rotaevaporated as before, theproduct extracted with DCM, centrifuged to remove suspended NaCl/NaI,and washed 3 times with H₂O. DCM was then removed once again, and theoily orange/brown product was taken up in 10 mL acetone and stored at−20° C. in the dark.

The acryloyl group was introduced in a manner identical to Eq1, usingacryloyl chloride (Cl—CO—CH═CH₂, Sigma Aldrich) instead.

The reaction of thiol groups with iodoacetyl groups proceeds accordingto:

The resultant HI (hydrogen iodide) is quenched by the presence of abase. The reaction of thiol groups with the acryloyl functionalityproceeds according to:

Neutral to slightly basic pH is desirable for achieving selectivitytowards thiols. Thus, all reactions were done with purified reducedlinear 3-Cys peptide pre-dissolved in 30% acetonitrile in water, in thepresence of 100 mM NH₄HCO₃ (˜pH 8). Peptide concentrations were between100 and 1000 mM, and scaffold concentrations were kept in slight excess(˜1.5-fold). This entailed addition of the appropriate volume of acetonescaffold stock solution. Scaffold concentrations in the acetone stockswere estimated from the initial quantity of precursor employed, assumingquantitiative conversion to the iodoacetylated or acrylated derivatives,and 20% losses during the workup.

Several 3-Cys peptides, of the following sequences, were tested(cysteines are underlined):

Pep48-3s1 (SEQ ID No. 8) SDDCVRFGWTCPTVMCG Pep48-58 (SEQ ID No. 9)SDDCVRFGWTCEPSLPGCG Pep48-37 (SEQ ID No. 10) SDCVRFGWTCSPGMVGCD

All iodoacetyl scaffolds reacted with any of the peptides very rapidly(conversion was usually complete within 10 minutes under theseconditions), as judged by mass spectrometry (MALDI). As an example, thereaction of TPEA(IAc3) ((R,R,R)-tris(2-phenyl)-ethanol aminetrisiodoacetate) with Pep48-58 was sampled after 5 min and analysed byMALDI.

The mass increase of 499 Da (due to addition of the scaffold, andelimination of 3 HI) correlates well with the theoretical expected value(500 Da, Table 1). Intermediate products (i.e. higher masses with aniodine atom still attached to the scaffold, while partially coupledelsewhere to the peptide) were not observed on any occasion. Thereaction with more constrained scaffolds (i.e. TOHPT(IAc3), see Table 1)was slower (˜1 hour). Intermediate coupling products were not observedon this occasion either (i.e. +1 HI, +2 HI).

A Pep48-37 derivative, where all Cys were replaced by Lys, did not showappreciable modification by THME(IAc3) after 2 hours—indicating goodselectivity of the iodoacetyl functionality for cysteines. The acrylatedscaffolds THME(Acr3) and TPBA(Acr3) coupled to the 3-Cys peptides aswell, albeit at a slower rate (˜<1 hr).

TABLE 1 List of Scaffolds synthesised and tested against Peptidescontaining 3 Cysteines. Name MW ^(a) Scaffold Precursors ^(b)Thiol-reactive Scaffold, final product ^(c) Chemistry THME(IAc3)Trishydroxy- methylethane trisiodoacetate 239

Iodoacetyl PE(IAc4) Pentaerythritol tetraiodoacetate 296

Iodoacetyl TEA(IAc3) Triethanolamine trisiodoacetate 272

Iodoacetyl TPEA(IAc3) (R,R,R)-tris (2-phenyl)- ethanolaminetrisiodoacetate 500

Iodoacetyl TPBA(IAc3) (Tris p-benzyl) amine trisiodoacetate Trisp-methyl- phenylamine trisiodoacetate 455

Iodoacetyl

TOHPT(IAc3) Tris o- hydroxyphenyl triazine trisiodoacetate 477

Iodoacetyl

THME(Acr3) Trishydroxy- methylethane trisacrylate 282^(d)

Acryloyl TPT(Acr3) Trispiperazine- triazine triacrylate 496^(d)

Acryloyl ^(a) The molecular weight corresponds to the mass addition to a3-Cys peptide assuming all 3 iodines and 3 hydrogens have been displacedfor any of the iodoacetyl-containing scaffolds. ^(b) Starting materialsemployed for the synthesis of the precursor scaffold (excluding thethiol-reactive functionality). ^(c) Final scaffold showing thethiol-reactive chemistry employed. On occasion more than one view of themolecule is shown (TBH/TOHBT-IAc3). ^(d)As the addition reaction ofacryloyl groups with thiols incurs no loss in mass, the MW indicatedrepresents that of the entire scaffold

Example 5 Use of Trishydroxymethylethane Trisiodoacetate (THME(IAc3))Cores

As described in Example 1, peptide conjugates were made by selectionagainst the protein MDM2. In the case of PEP48 the sequence of thesecond loop was randomized, and the phage repertoire subjected tofurther rounds of selection. This led to several conjugates withimproved binding affinity (see Example 4 for sequences of several suchPEP48 derivatives). One of these, the conjugate PEP48-3s1 had a bindingaffinity as measured by fluorescence anisotropy in the range 27 to 100nM.

The same peptide PEP48-3s1 was conjugated as above with THME(IAc3)instead of TBMB, as described in Example 4. The three iodoacetyl groupsare expected to react with up to three cysteine residues of a peptide;in this case the core is expected to impose a tetrahedral geometry onthe peptide, and lead to two stereoisomers. This is consistent with thetwo peaks of identical mass seen on purification of the conjugate withHPLC, which had different binding affinities when measured byfluorescence anisotropy (640 nM and 1600 nM).

Thus as expected the different geometry and packing imposed by theTHME(IAc3) core differs from that imposed by the TBMB core, and leads toan altered binding affinity.

Example 6 Making Core Variants: Synthetic Pathway to BenzoxazoleDerivatives

By way of illustration, a reagent to make cores, such as TBMB, can bechemically derivatized to make variant cores. The following exampleindicates two synthetic pathways that might be used to createbenzoxazole derivatives.

Koci et al, Bioorg Med Chem Lett, 2002, 12, 3275-3278.

2-mercaptobenzoxazole (5 mmol, 151 mg), dissolved in dry DMF (8 mL), isadded to a solution of sodium (5 mmol, 115 mg) in dry ethanol (2.5 mL).After 10 min of stirring at room temperature, TBMB (5 mmol, 1.785 g) isadded by portions and the resultant suspension is stirred for 6 hours.The reaction mixture is then poured into an ice bath and left overnight.The solid obtained is filtered off, washed with cold water (3×20 mL) andair-dried. The crude product is purified by flash chromatography usingEtOAc-hexane (20/80) as eluent system.

Heinen et al, Angewandte Chemie, 2000, 39, 806-809.

1,3,5-Tris(hydroxymethyl)benzene (11.9 mmol) and triphenylphosphine(11.9 mmol) are stirred under argon in 100 mL dry THF. The flask iscooled to 0° C. and tetrabromomethane (11.9 mmol) is added. The solutionis stirred at room temperature for 2.5 h and the reaction is monitoredthrough TLC. Triphenylphosphineoxide, which settled down, was filtered.The filtrate was evaporated to give a residue which was thenfractionated by flash chromatography using EtOAc as eluent to obtainbromomethyl-3,5-bis(hydroxymethyl)benzene.

2-mercaptobenzoxazole (5 mmol), dissolved in dry DMF (8 mL), is added toa solution of sodium (5 mmol) in dry ethanol (2.5 mL). After 10 min ofstirring at room temperature, bromomethyl-3,5-bis(hydroxymethyl)benzene(5 mmol) is added by portions and the resultant suspension is stirredfor 6 hours. The reaction mixture is then poured into an ice bath andleft overnight. The solid obtained is filtered off, washed with coldwater (3×20 mL) and air-dried. The crude product is purified by flashchromatography using EtOAc-hexane (20/80) as eluent system.

Benzoxazolyl-2′-thiomethyl-3,5-bis(hydroxymethyl)benzene (1.2 mmol) andtriphenylphosphine (1.2 mmol) are stirred under argon in 10 mL dry THF.The flask is cooled to 0° C. and tetrabromomethane (1.2 mmol) is added.The solution is stirred at room temperature for 2.5 h and the reactionis monitored through TLC. Triphenylphosphineoxide, which settled down,was filtered. The filtrate was evaporated to give a residue which wasthen fractionated by flash chromatography using EtOAc as eluent toobtain benzoxazolyl-2′-thiomethyl-3,5-bis(bromomethyl)benzene.

Such benzoxazole conjugates might be used according to the invention asfollows. The polypeptides of a first repertoire of polypeptide ligandscomprising two cysteine residues separated by a loop sequence conjugatedto dibromomethylbenzene, could be conjugated to the above product ofbenzoxazole and TBMB. This is expected to alter the conformationaldiversity of the first repertoire as the second repertoire ofpolypeptides will comprise an extra benzoxazole ring attached to thescaffold. In turn this may provide ligands selected from the secondrepertoire with additional constraints and binding contacts (leading toimproved affinity and/or specificity to target, and/or enhanced proteaseresistance).

The addition of other functions to the core for example moieties thatbind to serum albumin (for half-life extension), or guanidine moieties(for cell penetration), or enzyme inhibitors, or toxic drugs (for cellkilling) could also be used to alter the conformational diversity of arepertoire.

Example 7 Orthogonal Reactive Groups of the Scaffold

A peptide comprising the first loop of the kallikrein inhibitor PK15(with additional aspartic acid residues at the N- and C-terminus) wascoupled to TBMB and subsequently to propylargylamine to yield theproduct below:

The reaction details are as follows: PK15-L1DD ((Ac)-DCSDRFRNCD-(NH₂))(SEQ ID No. 11) (5 mg) in 600 μl water was treated with TCEP (60 μl) for30 mins and then HPLC purified (2 runs, eluted in ca 7 ml buffer, m/z1271). The buffer was treated with an equal volume of 100 mM ammoniumbicarbonate to pH ˜8 and TBMB (5 equivalents, 7 mg in 0.5 ml MeCN) andreaction left at room temperature. After 20 mins MALDI MS showedcompletion of reaction (m/z 1375) and the reaction quenched withpropargylamine (50 equivalents, 10 μl) and the reaction left overnight.The product was HPLC purified and lyophilised to a white powder (m/z1440, calc. 1440).

The propargylamine provides a scaffold reactive group that is orthogonalto the benzylic bromines of TBMB. It is capable of reacting by clickchemistry with an azido function within or attached to the polypeptide,and thereby creating a second polypeptide loop. Azido functions can beprovided by incorporation of azido amino acids (for exampleazido-tyrosine) into the polypeptide, or by using bifunctional linkers.

Furthermore the propargylamine is capable of reacting with otherpolypeptides; here we describe its use to couple to a second polypeptideligand to make a bifunctional ligand.

Firstly a peptide comprising the first loop of the MDM2 inhibitor PEP48(with additional aspartic acid residues at the N- and C-terminus) wascoupled to DBMB follows. Pep48-L1 DD ((H)-DGCVRFGWTCD-(NH₂) (SEQ ID No.12)) (5 mg) in 600 μl water was treated with TCEP (60 μl) for 30 minsand then HPLC purified (2 runs, eluted in ca 7 ml buffer, m/z 1258). Thebuffer was treated with an equal volume of 100 mM ammonium bicarbonateto pH ˜8 and DBMB (5 mg in 0.5 ml MeCN) and reaction left at roomtemperature. After 20 mins MALDI MS showed completion of reaction andproduct purified by HPLC, and lyophilised. The product was re-dissolvedin water (1 ml) and to this added 10 equivalents of the azide linker(see structure below, 4.5 mg in 100 μl 100 mM ammonium carbonate) andEDC (5 mg in 100 μl water) and reaction left overnight. MALDI MS showeddesired product (m/z 1471, calc. 1472), which was purified by HPLC.

The PK15-L1DD-TBMB-propargylamine conjugate (ca 1 mg, ca 0.7 μmole) wasthen dissolved in 0.5 ml water and to this added sodium ascorbate (50 μlof 25 μM in 200 mM NaCl), copper sulphate (25 μl of 2.5 μM in 200 mMNaCl) and the azide linker-modified Pep48-L1 DD-DBMB conjugate (above,ca 1 mg). MALDI MS showed product after 1 hr which was HPLC purified(m/z 2911, calc. 2910 for expected structure as below)

Example 8 Altering Reactive Groups of the Peptide

Earlier experiments had shown the reaction of cysteine with TBMB.Methionine was found to react with both TBMB and DBMB. A solution ofmethionine (1 mg/ml) in 1:1 acetonitrile:water and 25 mM ammoniumbicarbonate was treated with either DBMB or TBMB (1 mg/ml in MeCN).Reaction was monitored by LC-MS after 1 hr reaction. With DBMBmethionine showed a product m/z 334 (calc. 333 for single addition ofDBMB) and with TBMB a product of m/z 426 (calc. for single addition ofTBMB), see proposed product below:

When we altered the central cysteine of the PK15 peptide to methionine[sequence (H)-ACSDRFRNMPADEALCG-(OH) (SEQ ID No. 13)] and reacted itwith TBMB, we observed two peaks on HPLC with identical mass, with mass(1884) as expected to a conjugate of the TBMB through both cysteines andthe single methionine. Both products inhibited the activity ofkallikrein, but with 1050 values above 10 micromolar. Thus the changingof cysteine for methionine has a dramatic effect on the binding affinitypresumably due to altered packing interactions from the additionalmethylene and methyl groups, and/or geometry of bonds around themethionine sulphur atom, and/or additional positive charge.

All publications mentioned in the above specification are hereinincorporated by reference. Various modifications and variations of thedescribed aspects and embodiments of the present invention will beapparent to those skilled in the art without departing from the scope ofthe present invention. Although the present invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are apparent tothose skilled in the art are intended to be within the scope of thefollowing claims.

1. A method for altering the conformation of a first polypeptide ligandor group of polypeptide ligands, each polypeptide ligand comprising atleast two reactive groups separated by a loop sequence covalently linkedto a molecular scaffold which forms covalent bonds with said reactivegroups, to produce a second polypeptide ligand or group of polypeptideligands, comprising assembling said second ligand or group of ligandsfrom the polypeptide(s) and structural scaffold of said first ligand orgroup of ligands, incorporating one of: (a) altering at least onereactive group; or (b) altering the nature of the molecular scaffold; or(c) altering the bond between at least one reactive group and themolecular scaffold; or (d) any combination of (a), (b) or (c).
 2. Themethod of claim 1, wherein the reactive group comprises a sulphur atom.3. The method of claim 2, wherein the reactive group is a cysteine or amethionine.
 4. The method of claim 2, wherein the reactive group isaltered to cysteine, selenocysteine or methionine.
 5. The method ofclaim 1, wherein the altered molecular scaffold is asymmetric.
 6. Themethod of claim 5, wherein the molecular scaffold comprises two or morescaffold reactive groups which are capable of forming covalent bondswith the reactive groups on the polypeptides, and said two or morereactive groups are not identical.
 7. The method of 6, wherein one ofsaid scaffold reactive groups is orthogonal.
 8. The method of claim 1,wherein the scaffold reactive groups are selected from benzylic halides,α-halocarboxylic acids and acryloyl moieties.
 9. The method of claim 1,wherein at least one bond between the molecular scaffold and thepolypeptide are chemically modified after assembly of the polypeptideand the molecular scaffold.
 10. The method of claim 9, wherein at leastone thio-ether linkage between the polypeptide and the molecularscaffold is oxidised to form a sulphoxide or a sulphone.
 11. The methodof claim 1, wherein the first group of polypeptide variants is a firstrepertoire of polypeptide variants.
 12. The method of claim 1, whereinthe second group of polypeptide variants is a second repertoire ofpolypeptide variants.
 13. A method for generating a group or repertoireof diverse polypeptide ligands from a first polypeptide ligandcomprising at least two reactive groups separated by a loop sequencecovalently linked to a molecular scaffold which forms covalent bondswith said reactive groups, comprising assembling a group or repertoireof ligands from the polypeptide and scaffold of said first ligand orgroup of ligands, and incorporating one of: a) altering at least onereactive group; or b) altering the nature of the molecular scaffold; orc) altering the bond between at least one reactive group and themolecular scaffold; or d) any combination of (a), (b) or (c); or e)modifying the sequence of the polypeptide, in combination with any oneof (a) to (d).
 14. A method for increasing the conformational diversityof a first repertoire of polypeptide ligands, comprising a plurality ofpolypeptides comprising at least two reactive groups separated by a loopsequence covalently linked to a molecular scaffold which forms covalentbonds with said reactive groups, comprising assembling a secondrepertoire of peptide ligands from the polypeptides and scaffold of saidfirst repertoire, incorporating one of: (a) altering at least onereactive group; or (b) altering the nature of the molecular scaffold; or(c) altering the bond between at least one reactive group and themolecular scaffold; or (d) any combination of (a), (b) or (c). (e)modifying the sequence of the polypeptide, in combination with any oneof (a) to (d).
 15. A method according to claim 14, wherein the secondrepertoire is assembled from the polypeptides of the first repertoire,and at least two structurally diverse molecular scaffold species.
 16. Amethod for providing a polypeptide ligand comprising a polypeptidecovalently linked to a molecular scaffold at three or more amino acidresidues, comprising the steps of: a. providing a first repertoire ofpolypeptides; b. conjugating said polypeptides to a molecular scaffoldwhich binds to the polypeptides at two or more amino acid residues, toform a first repertoire of polypeptide conjugates; c. screening saidfirst repertoire for binding against a target, and selecting members ofthe first repertoire which bind to the target; d. introducing furthervariation into the polypeptide ligands, in accordance with claim 1,yielding a second repertoire of polypeptide conjugates; and e. screeningsaid second repertoire for improved binding to the target.
 17. A methodfor selecting a peptide ligand having increased protease resistance,comprising the steps of: a. providing a first repertoire ofpolypeptides; b. conjugating said polypeptides to a molecular scaffoldwhich binds to the polypeptides at two or more amino acid residues, toform a first repertoire of polypeptide conjugates; c. screening saidfirst repertoire for binding against a target, and selecting members ofthe first repertoire which bind to the target; d. introducing furthervariation into the polypeptide ligands, in accordance with the firstaspect of the invention set forth above, yielding a second repertoire ofpolypeptide conjugates; e. subjecting the second repertoire to selectionfor protease resistance; and f. optionally, screening said secondrepertoire for binding to the target.
 18. The method of claim 17,wherein one or more of the groups or repertoires of polypeptides isencoded by one or more libraries of nucleic acid molecules.
 19. Themethod of claim 18, wherein the screening of said repertoires isperformed using a genetic display system.
 20. A method according toclaim 19, wherein the genetic display system is phage display.
 21. Arepertoire of peptide ligands, when produced by a method according toclaim
 17. 22. The method of claim 17, wherein any of the groups orrepertoires is made from synthetic peptides.
 23. The method of claim 22wherein the second group or repertoire is made from synthetic peptides.24. The method of claim 22, wherein said synthetic peptides are arrayedon solid phase.
 25. A group of peptide ligands, when produced by themethod according to claim
 24. 26. A polypeptide conjugate produced bythe method of 20, comprising a polypeptide comprising at least tworeactive groups each separated by a loop sequence, covalently linked toa molecular scaffold comprising at least two scaffold reactive groupswhich form covalent bonds with said reactive groups, wherein at leasttwo of said scaffold reactive groups are different.
 27. A polypeptideligand according to claim 26, which has a molecular weight of less than3000 Dalton.
 28. A polypeptide ligand according to claim 26, wherein thelength of the polypeptide loops subtended between any two adjacentpoints of attachment of the polypeptide to the molecular scaffold isbetween 0 and 9 amino acids, and the length of the polypeptide sequencebetween the first and last of said points of attachment is less than 27amino acids.